MX2011004557A - Formulations of single domain antigen binding molecules. - Google Patents

Abstract

The invention relates to formulations of single domain antigen binding molecules, <i>e.g.</i>, nanobody molecules, in particular formulations of TNF-binding nanobody molecules. The single domain antigen binding molecules can include one or more single binding domains that interact with, e.g., bind to, one or more target proteins. The formulations are useful,<i> e.g.</i>, as pharmaceutical formulations. Method of preparing, and using the formulations described herein, to treat, <i>e.g.</i>, TNF-associated disorders, are also disclosed.

Description

FORMULATIONS OF ANTIGEN BINDING MOLECULES SINGLE DOMAIN CROSS REFERENCE WITH RESPECT TO RELATED REQUESTS The present application claims priority with respect to document Serial Number US 61 / 109,474, filed on October 29, 2008, whose content is incorporated in this document by reference in its entirety.

REFERENCE TO LIST OF SEQUENCES The present application contains a Sequence List that has been submitted by EFS-Web and is incorporated herein by reference in its entirety. This ASCII copy, created on October 27, 2009, it is called W22373WO.txt, and it has a size of 8,343 bytes.

BACKGROUND OF THE INVENTION Advances in biotechnology have made it possible to produce a variety of proteins for pharmaceutical applications using recombinant DNA techniques. Because proteins tend to be larger and More complex than traditional organic and inorganic drugs, the formulation of such proteins presents special problems. For a protein to remain biologically active, the formulation must retain the conformational integrity of at least one core sequence of the amino acids in the proteins, while protecting the multiple functional groups of the protein from degradation. Degradation pathways for proteins can involve chemical instability (ie, any of the processes involving protein modification by bonding or cleavage resulting in a new chemical entity) or physical instability (i.e., changes in structure). of higher order of the protein). Chemical instability can result, for example, in deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide bridge exchange. Physical instability can result from, for example, denaturation, aggregation, precipitation or adsorption. Three common degradation pathways in proteins are aggregation, deamidation and oxidation of proteins (Cleland et al Critical Review in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993)).

Freeze drying is a technique commonly used to conserve proteins used to remove water from the preparation of the protein of interest. Freeze drying or lyophilization is a process by which the material to be dried is frozen first and then the frozen or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in the pre-formulations lyophilized to enhance the stability during the freeze drying process and / or to improve the stability of the lyophilized product during storage (Pikal, M. Biopharm 3 (9) 26-30 (1990) and Arakawa et al., Pharm. Res. 8 (3): 285-291 (1991)).

Therefore, there is still a need to develop protein formulations, particularly for subcutaneous administration, whose preservation and delivery are stable for a long time.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to formulations of single domain antigen-binding molecules (also referred to herein as "UADS molecules" (eg, nanobody molecules, particularly formulations of TNF-binding nanobody molecules)). The UADS molecule may include one or more simple antigen-binding domains that interact with, for example, binding to, one or more target proteins. The formulations are useful, for example, as pharmaceutical formulations, for administration to a subject, for example, a human. Also disclosed are methods for preparing and using the formulations described herein, for treating or preventing, for example, disorders associated with TNF.

[Note: Nanobody ™ and Nanobodies ™ are registered trademarks of Ablynx N. V] Accordingly, in one aspect, the present invention relates to a formulation that includes (a) a UADS molecule, e.g., a nanobody molecule (e.g., a TNF-binding nanobody molecule); (b) a lyoprotectant; (c) (optionally) a surfactant; (d) (optionally) a bulking agent; (e) (optionally) a tonicity adjusting agent; (f) (optionally) a stabilizer; (g) (optionally) a preservative, and (h) a buffer, such that the pH of the formulation is from about 5.0 to 7.5. In some embodiments, the formulation is a liquid formulation, a lyophilized formulation, a reconstituted lyophilized formulation, an aerosol formulation, or a bulk storage formulation (e.g., frozen bulk storage formulation). In some embodiments, the formulation is administered to a subject by injection (eg, subcutaneous, intravascular, intramuscular or intraperitoneal) or by inhalation.

In some embodiments, the UADS molecule, e.g., the nanobody molecule (e.g., the TNF-binding nanobody molecule), in the formulation is in a concentration of about 0.5 mg / ml to about 350 mg / ml, about 0.5 mg / ml to about 300 mg / ml, about 0.5 mg / ml to about 250 mg / ml, about 0.5 mg / ml to about 150 mg / ml, about 1 mg / ml to about 130 mg / ml, about 10 mg / ml to approximately 130 mg / ml, approximately 50 mg / ml to approximately 120 mg / ml, about 80 mg / ml to about 120 mg / ml, about 88 mg / ml to about 100 mg / ml or about 10 mg / ml, about 25 mg / ml, about 50 mg / ml, about 80 mg / ml, about 100 mg / ml, approximately 130 mg / ml, approximately 150 mg / ml, approximately 200 mg / ml, approximately 250 mg / ml or approximately 300 mg / ml.

In other embodiments, the lyoprotectant of the formulation is a sugar, for example, sucrose, sorbitol or trehalose. For example, the lyoprotectant may be sucrose, sorbitol or trehalose at a concentration of from about 2.5% to about 10%, about 5% to about 10%, about 5% to about 8% or about 4%, about 4.5%, about 5%. %, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5% or about 9% (weight / volume).

In still other embodiments, the buffer in the formulation is a histidine buffer at a concentration of about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 20 mM or about 10 mM, about 20 mM or about 30 mM. In other embodiments, the buffer in the formulation is a Tris buffer present at a concentration less than about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 20 mM, or about 10 mM, about 20 mM or about 30 mM. The pH of the formulation buffers is generally between about 5 and 7. In some specific embodiments, the pH of the formulation buffer is from about 5 to about 7.5, about 5.5 to about 7.2. For example, the pH of the buffer may be about 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7.

In some embodiments, the formulation (optionally) includes a surfactant at a concentration of about 0.001% to 0.6%, for example, about 0.01% to 0.6%, about 0.1% to 0.6%, about 0.1% at 0.5%, approximately 0.1% to 0.4%, approximately 0.1% to 0.3%, approximately 0.1% to 0.2% or approximately 0.01% to 0.02%. In some cases, the formulation contains more than 0% and up to about 0.6% (eg, about 0.1% to 0.2% polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate -85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleastate, or a combination thereof In specific embodiments, the formulation contains approximately 0.001%, 0.002%, 0.003%, 0.004%, 0. 005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01% to 0.02%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% , 0.1% to 0.2%, 0.1%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% or 0.2% polysorbate-80. Alternatively, the formulation may include poloxamer-188 to about 0.01% to 0.6%, about 0.1% to 0.6%, about 0.1% to 0.5%, about 0.1% to 0.4%, about 0.1% to 0 , 3% or approximately 0.1% to 0.2%.

In some embodiments, the formulation (optionally) includes a bulking agent, eg, glycine, at a concentration of about 10 to about 200 mM, about 25 to about 175 mM, about 50 to about 150 mM, about 75 to approximately 125 mM or approximately 100 mM.

In other embodiments, the formulation (optionally) further includes a tonicity adjusting agent, for example, a molecule that makes the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride.

In other additional embodiments, the formulation (optionally) further includes a stabilizer, e.g., a molecule that, when combined with a protein of interest (e.g., the UADS molecule) prevents or substantially reduces instability chemistry and / or physics of the protein of interest in storage form, lyophilized or liquid. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine and arginine hydrochloride. In some embodiments, the formulations include a stabilizer in one or more of the following ranges: sucrose from about 1% to about 12% (e.g., about 5%, about 7.5%, about 8%, or about 0%); sorbitol from about 1% to about 7% (eg, about 3%, about 4%, about 5%); inositol from about 1% to about 5%; glycine of about 10 mM to about 125 mM (eg, about 25 mM to 100 mM, about 80 mM, about 90 mM or about 100 mM); Sodium chloride of about 10 mM to 150 mM (eg, about 25 mM to 100 mM, about 55 mM); methionine from about 10 mM to about 100 mM (eg, about 10 mM, about 20 mM, about 100 mM); arginine from about 10 mM to about 125 mM (eg, about 25 mM to about 120 mM or about 100 mM); arginine hydrochloride from about 10 mM to about 70 mM (eg, about 10 mM to about 65 mM or about 55 mM).

In other embodiments, the formulation may additionally include methionine, at a concentration of about 10 to about 200 mM, about 25 to about 175 mM, about 50 to about 150 mM, about 75 to about 125 mM, or about 100 mM. .

In one embodiment, a component of the formulation can act as one or more of a lyoprotectant, a tonicity adjusting agent and / or a stabilizer. For example, depending on the concentration of a component, for example sucrose, it can serve as one or more of a lyoprotectant, a tonicity adjusting agent and / or a stabilizer. In other embodiments when several of the components in a formulation are needed, different components are used. For example, when the formulation requires a lyoprotectant, a tonicity adjusting agent and a stabilizer, different components are used (for example, sucrose, glycine and inositol can be used in combination resulting in a combination of a lyoprotectant, a tonicity adjusting agent. and a stabilizer, respectively).

In one embodiment, the formulation includes (a) a UADS molecule, e.g., a nanobody molecule (e.g., a TNF-binding nanobody molecule) at a concentration of about 0.5 to about 300 mg / ml, e.g. , approximately 1 mg / ml, approximately 10 mg / ml, approximately 25 mg / ml, approximately 50 mg / ml, approximately 80 mg / ml, approximately 88 mg / ml, approximately 100 mg / ml, approximately 18 mg / ml, approximately 130 mg / ml, approximately 150 mg / ml or approximately 250 mg / ml; (b) sucrose at a concentration of about 5% to about 10%, for example, about 5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 10%; (c) polysorbate-80 at a concentration of from about 0 to about 0.6%, eg, 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 0.6%; (d) (optionally) glycine at a concentration of from about 0 to about 100 m, for example, 100 mM; (e) (optionally) methionine at a concentration of about 0 to about 100 mM, eg, 100 mM; and (f) a histidine buffer (at a concentration of approximately 10 mM to approximately 20 mM) or a Tris buffer (at a concentration approximately 20 mM), such that the pH of the formulation is from about 5.0 to 7.5, for example , 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7.

In one embodiment, the formulation is a liquid formulation. In a representative embodiment the liquid formulation includes a) a UADS molecule, for example, a nanobody molecule (e.g., a TNF-binding nanobody molecule) at a concentration of about 10 to about 150 mg / ml, e.g. 25 mg / ml, approximately 50 mg / ml, approximately 80 mg / ml, approximately 88 mg / ml, approximately 100 mg / ml, approximately 118 mg / ml, approximately 130 mg / ml; (b) sucrose at a concentration of from about 5% to about 10%, for example from about 7% to about 8%, e.g., 7.5% or sorbitol from about 1% to about 7% (e.g., about 3%, about 4%, about 5%) (c) polysorbate-80 at a concentration of about, for example, about 0.01% to 0.02% (eg, 0.01%); (d) (optionally) glycine at a concentration of about 0 to about 100 mM, eg, 100 mM; (e) (optionally) methionine at a concentration of about 0 to about 100 mM, eg, 100 mM; and (f) a histidine buffer (at a concentration of about 10 mM to about 20 mM) or a Tris buffer (at a concentration of about 20 mM), so that the pH of the formulation is about 5 to 7.5, for example, 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7. The liquid formulation can be present in a preparation article, such as a device, a syringe or a vial with instructions for its use. In some embodiments, the syringe or vial is composed of glass, plastic or a polymeric material, such as cyclic olefin polymer or copolymer. In other embodiments, the formulation may be present in an injectable device (e.g., an injectable syringe, e.g., a pre-filled syringe). The syringe can be adapted for individual administration, for example, as a single vial system that it includes an autoinjector (for example, a pen injector device) and / or instructions for its use. The formulation can be administered to a subject, for example, a patient by injection, e.g., peripheral administration (e.g., subcutaneous, intravascular, intramuscular or intraperitoneal administration).

In other embodiments, the formulation is a lyophilized formulation. In a representative embodiment, the lyophilized formulation includes a) a UADS molecule, e.g., a nanobody molecule (e.g., a TNF-binding nanobody molecule) at a concentration of about 10 to about 150 mg / ml, e.g. about 25 mg / ml, about 50 mg / ml, about 80 mg / ml, about 88 mg / ml, about 100 mg / ml, about 118 mg / ml, about 130 mg / ml; (b) sucrose at a concentration of about 5% to about 10%, for example, about 4% to about 7%, e.g., 5%; (c) polysorbate-80 at a concentration of about, for example, 0.01% to 0.02% (eg, 0.01%); (d) (optionally) glycine at a concentration of about 0 to about 100 mM, eg, 100 mM; (e) (optionally) methionine at a concentration of about 0 to about 100 mM, eg, 100 mM; and (f) a histidine buffer (at a concentration of approximately 10 mM to approximately 20 mM, for example, approximately 20 mM), or a Tris buffer (at a concentration approximately 20 mM), so that the pH of the formulation is from about 5 to 7.5, for example, 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7. The lyophilized formulation can be reconstituted by mixing the lyophilizate with a suitable aqueous composition.

In still other embodiments, the formulation is a bulk storage formulation. In a representative embodiment, the bulk storage formulation includes a) a UADS molecule, e.g., a nanobody molecule (e.g., a TNF-binding nanobody molecule) at a concentration of about 80 mg / ml to 300 mg / ml, for example, about 150 mg / ml, about 175 mg / ml, about 200 mg / ml, about 250 mg / ml, about 275 mg / ml or about 300 mg / ml; (b) sucrose at a concentration of from about 5% to about 10%, for example about 4% to about 8%, for example, 5% or 7.5%; (c) polysorbate-80 at a concentration of about, for example, 0.01% to 0.02%; (d) (optionally) glycine at a concentration of about 0 to about 100 mM, eg, 100 mM; (e) (optionally) methionine at a concentration of about 0 to about 100 mM, eg, 100 mM; and (f) a histidine buffer (at a concentration of approximately 10 mM to approximately 20 mM) or a Tris buffer (at a concentration approximately 20 mM), such that the pH of the formulation is from about 5 to 7.5, for example , 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7. The bulk storage formulation can be frozen. In some embodiments, the bulk preservation formulation can be prepared on a large scale, for example, more than 10 liters, 50 liters, 100, 150, 200 or more liters.

In some embodiments, the UADS molecule, for example, the nanobody molecule (e.g., the TNF-binding nanobody molecule) of the formulation includes one or more single binding domains (e.g., one or more nanobodies). For example, the nanobody molecule may comprise or consist of a polypeptide, for example, a single-chain polypeptide comprising at least one immunoglobulin variable domain (including one, two or three complementarity determining regions (CDRs)). Examples of UADS molecules include molecules naturally devoid of light chains (e.g., nanobodies, VHH or antibodies derived from camelids). Said UADS molecules can be derived or obtained from camelids such as camel, llama, dromedary, alpaca and guanaco. In other embodiments, the UADS molecule may include single domain molecules including, but not limited to, other single domain molecules of natural origin, such as shark single domain polypeptides (IgNAR); and single domain frameworks (for example, fibronectin frameworks). Single domain molecules can be derived from shark.

In one embodiment, the UADS molecule of the formulation is a single-chain polypeptide comprising one or more single domain molecules. In embodiments, the nanobody molecule is monovalent or multivalent (eg, bivalent, trivalent or tetravalent). In other embodiments, the nanobody molecule is monospecific or multispecific (by example, bispecific, trispecific or tetraespecific). The UADS molecule may comprise one or more single domain molecules that are recombinant, CDR grafted, humanized, camelized, deimmunized and / or generated in vitro (eg, selected by phage display). For example, the UADS molecule can be a single-chain fusion polypeptide comprising one or more single domain molecules that bind to one or more target antigens. Typically, the target antigen is a mammalian protein, for example, a human. In certain embodiments, the UADS molecule binds to a serum protein, for example, human serum proteins selected from one or more of serum albumin (human serum albumin (HSA)), fibrin, fibrinogen or transferrin.

In an exemplary embodiment, the UADS molecule of the formulation is a bispecific, trivalent molecule composed of a single-stranded polypeptide fusion of two single domain molecules (eg, two variable camelid regions) that bind to a target antigen, by example, tumor necrosis factor a (TNF-a), and a single domain molecule (e.g., a variable camelid region) that binds to a serum protein, e.g., ASH. The single domain molecules of the UADS molecule can be arranged in the following order from the N- to C-terminus: single domain molecule binding to TNFa - single domain molecule binding to ASH - single domain binding molecule TNFa. It will be appreciated that any order or combination of the molecules of Single domain against one or more targets can be formulated as described herein.

In one embodiment, the UADS molecule of the formulation is referred to herein as "ATN-103," comprising or consisting of the amino acid sequence shown in Figure 30 (SEQ ID NO: 1), or an amino acid sequence substantially identical to it (for example, an amino acid sequence of at least 85%, 90%, 95% or more identical to or having up to 20, 15, 10, 5, 4, 3, 2, 1 amino acid changes (per Examples, deletions, insertions or substitutions (e.g., conservative substitutions) with respect to the amino acid sequence shown in Figure 30.) Examples of additional trivalent bispecific nanobody molecules that can be formulated as described herein include TNF24, TNF25, TNF26, TNF27, TNF28, TNF60 and TNF62 disclosed in Table 29 of WO 2006/122786.

In some embodiments, at least one of the single domain molecule of the UADS molecule of the formulation binds to TNFa, includes one, two or three CDRs having the amino acid sequence: DYWMY (SEQ ID NO: 2) (CDR1 ), EINTNGLITKYPDSVKG (SEQ ID NO: 3) (CDR2) and / or SPSGFN (SEQ ID NO: 4) (CDR3), or having a CDR that differs by less than 3, 2 or 1 amino acid substitutions (e.g. conservative substitutions) of one of said CDRs. In other embodiments, the single domain molecule comprises a variable region having the amino acid sequence from about amino acids 1 through 115 of the Figure 30 or an amino acid sequence substantially identical thereto, (for example, an amino acid sequence of at least 85%, 90%, 95% or more identical to, or having up to 20, 15, 10, 5, 4 , 3, 2, 1 amino acid changes (e.g., deletions, insertions or substitutions (e.g., conservative substitutions) with respect to the amino acid sequence shown in Figure 30.) In embodiments, the single domain binding molecule TNFa has one or more biological activities of the single-domain TNFa-binding antibody molecule shown in Figure 30. For example, the single-domain TNFa-binding molecule binds to the same or a similar epitope as the epitope recognized by the single-domain TNFa-binding molecule shown in Figure 30 (e.g., binds to TNFa in its trimeric form, binds to the TNFa site by contacting the TNF receptor, binds to an epitope on the TNFa trimer understands Gln in the position 88 and Lys at position 90 of the first TNF monomer (monomer A), and Glu at position 146 of the second TNF monomer (monomer B), or an epitope as disclosed in WO 06/122786). In another embodiment, the single-domain TNFa-binding molecule has an activity (e.g., binding affinity, dissociation constant, binding specificity, inhibitory activity with respect to TNF) similar to any of the single binding domain molecule to TNFα disclosed in WO 06/122786.

In other embodiments, the TNFα binding nanobody molecule comprises one or more of the nanobodies disclosed in WO 2006/122786. For example, the TNFα binding nanobody molecule can be a trivalent, bivalent, monovalent TNFα-binding nanobody molecule disclosed in WO 2006/122786. Exemplary TNFa binding nanobodies include but are not limited to, TNF1, TNF2, TNF3, humanized forms thereof (eg, TNF29, TNF30, TNF31, TNF32, TNF33). Additional examples of monovalent TNF [alpha] binding nanobodies are disclosed in Table 8 of WO 2006/122786. Exemplary bivalent TNFa-binding nanobody molecules include, but are not limited to, TNF55 and TNF56, which comprise two TNF30 nanobodies linked by peptide linkage to form a single fusion polypeptide (disclosed in WO 2006/122786). Further examples of bivalent TNFα-binding nanobody molecules are disclosed in Table 19 of WO 2006/122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, at least one of the single domain molecule of the UADS molecule of the formulation binds ASH includes one, two, or three CDRs having the amino acid sequences: SFGMS (SEQ ID NO: 5) (CDR1) , SISGSGSDTLYADSVKG (SEQ ID NO: 6) (CDR2) and / or GGSLSR (SEQ ID NO: 7) (CDR3), or having a CDR that differs by less than 3, 2 or 1 amino acid substitutions (e.g. conservative) of one of said CDRs. In other embodiments, the molecule of single domain comprises a variable region having the amino acid sequence of about 125 to 239 amino acids of Figure 30 (SEQ ID NO: 1), or an amino acid sequence substantially identical thereto (eg, an amino acid sequence of less 85%, 90%, 95% or more of identity or you have up to 20, 15, 10, 5, 4, 3, 2, 1 amino acid changes (e.g., deletions, insertions or substitutions (e.g., conservative substitutions) with respect to the amino acid sequence shown in Figure 30 (SEQ ID NO: 1) In embodiments, the single ASH binding domain molecule has one or more biological activities of the ASH-binding single domain molecule shown in Figure 30 (SEQ ID NO: 1). Single ASH-binding domain binds to the same or a similar epitope as the epitope recognized by the ASH-binding single domain molecule shown in Figure 30 (SEQ ID NO: 1) In another embodiment, the domain molecule simple binding to ASH has an activity (eg, binding affinity, dissociation constant, binding specificity) similar to any of the single domain ASH binding domain molecule disclosed in WO 06/122786.

In other embodiments, the ASH-binding UADS molecule comprises one or more of the nanobodies disclosed in WO 2006/122786. For example, the ASH-binding UADS molecule can be a trivalent, bivalent, monovalent ASH-binding nanobody molecule disclosed in WO 2006/122786. In other embodiments, the UDSS binding molecule to ASH can be a monospecific molecule or multispecific that has at least one of the binding specificities that bind to ASH. Exemplary TNF binding nanobodies include, but are not limited to, ALB1, humanized forms thereof (eg, ALB6, ALB7, ALB8, ALB9, ALB10), disclosed in WO 06/122786.

In other embodiments, two or more of the single domain molecules of the UADS molecules are fused, with or without a linking group, such as a genetic or polypeptide fusion. The linking group can be any linking group evident to those skilled in the art. For example, the linking group can be a biocompatible polymer with a length of 1 to 100 atoms. In one embodiment, the linking group includes or consists of polyglycine, poly-serine, polylysine, polyglutamate, polyisoleucine or polyarginine moieties or a combination thereof. For example, the polyglycine or polyserin linkers may include at least five, seven, eight, nine, ten, twelve, fifteen, twenty, thirty, thirty-five and forty glycine and serine residues. The linkers that can be used include Gly-Ser repeats, for example, (Gly) 4-Ser (SEQ ID NO: 8) repeats of one, two, three, four, five, six, seven or more repeats. In embodiments, the linker has the following sequences: (Gly) 4-Ser- (Gly) 3-Ser (SEQ ID NO: 9) or ((Gly) 4-Ser) n (SEQ ID NO: 10), in the that n is 4, 5 or 6.

The formulations of the present invention can include a UADS molecule that is modified by, for example, covalently or non-covalently associating a second residue. For example, the nanobody molecule can be covalently bound to a suitable polymer pharmacologically acceptable, such as poly (ethylene glycol) (PEG) or a derivative thereof (such as methoxypoly (ethylene glycol) or mPEG). Examples of pegylated nanobody molecules are disclosed as TNF55-PEG40, TNF55-PEG60, TNF56-PEG40 and TNF56-PEG60 in WO 06/122786.

In another embodiment, the formulations of the present invention are stable for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months), at a temperature of about 2 ° C to about 25 ° C (for example, approximately 4 ° C or 25 ° C). In some embodiments, the integrity of the UADS molecule is maintained after storage in the formulation for at least 3, 6, 9, 12 months (eg, at least 24, 30, 36 months), at a temperature of about 2. ° C at approximately 25 ° C (for example, approximately 4 ° C or 25 ° C). For example, the UADS molecule in the formulation retains at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or up to 100% of a biological activity, for example, binding activity of the UADS molecule after storage at a temperature of about 2 ° C to about 25 ° C (e.g., about 4 ° C or 25 ° C). In some embodiments, the formulation includes high molecular weight (APM) species of less than 10%, 9%, 5%, 4%, 3%, 2%, 1% or less after storage in the formulation for at least 3 , 6, 9, 12 months (for example, at least 24, 30, 36 months), at a temperature of about 2 ° C to about 25 ° C (for example, about 4 ° C or 25 ° C). In other embodiments, the formulation includes low molecular weight (BPM) species of less than 10%, 9%, 5%, 4%, 3%, 2%, 1% or less after storage in the formulation for at least 3, 6, 9, 12 months (for example, at least 24, 30, 36 months), at a temperature of about 2 ° C to about 25 ° C (for example, approximately 4 ° C or 25 ° C). In still other embodiments, the formulation includes acid species of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage in the formulation for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months), at a temperature of about 2 ° C to about 25 ° C (e.g., about 4 ° C or 25 ° C) ). In still other embodiments, the formulation includes basic species of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less after storage in the formulation for at least 3, 6, 9, 12 months (e.g., at least 24, 30, 36 months), at a temperature of about 2 ° C to about 25 ° C (e.g., about 4 ° C or 25 ° C) ). The APM, BPM, acidic and basic species can be detected in the formulations using conventional techniques such as high performance liquid chromatography by size exclusion (HPLC-SEC) and the like described herein.

In some embodiments, after the reconstruction of the lyophilized UADS formulation, the formulation retains at least 80%, 90%, 95% or more of the UADS structure compared to the formulation prior to lyophilization. The UADS structure is determined, for example, by binding assay, bioassay, or the proportion of APM species with respect to BPM species.

The formulations of the invention may also include a second agent, for example, a second therapeutic or pharmacologically active active agent that is useful in the treatment of a disorder associated with TNF-a, for example, inflammatory or autoimmune disorders, including, but not limited to, without limitation, rheumatoid arthritis (RA) (eg, moderate to severe rheumatoid arthritis), arthritic states (eg, psoriatic arthritis, juvenile idiopathic polyarticular arthritis (JIA), ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease inflammatory bowel disease and / or multiple sclerosis For example, the second agent may be an anti-TNF antibody or a TNF-binding fragment thereof, wherein the second TNF antibody binds to a different epitope than the molecule of TNF binding UADS of the formulation Other non-limiting examples of agents that can be co-formulated with the UADS TNF binding molecule include, eg Without limitation, a cytokine inhibitor, a growth factor inhibitor, an immunosuppressant, an anti-inflammatory agent, a metabolic inhibitor, an enzyme inhibitor, a cytotoxic agent, a cytostatic agent. In one embodiment, the additional agent is a conventional treatment for arthritis, including, but not limited to, non-steroidal anti-inflammatory agents. (NSAID); corticosteroids, which include prednisolone, prednisone, cortisone, and triamcinolone; and disease-modifying antirheumatic drugs (FARMD), such as methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine, leflunomide (Arava®), tumor necrosis factor inhibitors, including etanercept (Enbrel®), infliximab (Remicade®) (with or without methotrexate), and adalimumab (Humira®), antibody anti-CD20 (eg, Rituxan®), soluble interleukin-1 receptor, such as anakinra (Kineret®), gold, minocycline (Minocin®), penicillamine and cytotoxic agents, including azathioprine, cyclophosphamide and cyclosporin. Said combination therapies can advantageously use lower dosages of the therapeutic agents administered, thus avoiding possible toxicities or complications associated with the various monotherapies.

Following the guidance provided herein, combinations of excipients and / or alternative secondary therapeutic agents can be identified and assayed.

In yet another embodiment, the formulations described herein are suitable for administration to a subject, for example, a human subject (e.g., a patient having a disorder associated with TNFa). The formulation can be administered to the subject by injection (eg, subcutaneous, intravascular, intramuscular or intraperitoneal) or by inhalation.

In another aspect, the invention features a process or process for preparing the formulations described herein. The procedure or process includes: expressing the UADS molecule in a cell culture; purifying the UADS molecule, for example, by passing the UADS molecule through at least one chromatographic purification step, ultrafiltration / diafiltration steps; adjusting the concentration of the UADS molecule, for example, to about 10 to 250 mg / ml in a formulation containing a lyoprotectant, a surfactant and a buffer as described herein, for example, sucrose at a concentration of about 5% to about 10%, for example, about 5%, about 0%; polysorbate-80 at a concentration of about 0 to about 0.02%, eg, 0.01%, 0.02%m. ; (optionally) glycine at a concentration of about 0 to about 100 mM, for example, 100 mM; (optionally) methionine at a concentration of about 0 to about 100 mM, eg, 100 mM; and (f) a Histidine (at a concentration of about 10 to about 20 mM) or a Tris buffer (at a concentration of about 20 mM), such that the pH of the formulation is about 5 to 7.5, e.g. 5, 5.5, 5.8-6.1, 6, 6.1, 6.5 OR 7.

In another aspect, the invention features a process or process for preparing a reconstituted formulation containing a UADS molecule, for example, a UADS TNF-binding molecule as described herein. The method includes: lyophilizing a mixture of a UADS molecule, a lyoprotectant, a surfactant and a buffer, thereby forming a lyophilized mixture; and reconstitute the lyophilized mixture in a diluent, thereby preparing a formulation as described herein. In one embodiment, the formulation includes (a) a UADS molecule, for example, a nanobody TNF binding molecule at a concentration of about 0.5 to about 200 mg / ml, eg, at about 1 mg / ml, about 50 mg / ml, about 80 mg / ml, about 88 mg / ml, about 100 mg / ml, about 118 mg / ml; (b) sucrose at a concentration of about 5% to about 10%, for example, about 5%, about 10%; (c) polysorbate-80 at a concentration of from about 0 to about 0.02%, for example, 0.01%, 0.02%; (d) (optionally) glycine at a concentration of about 0 to about 00 mM, eg, 100 mM; (e) (optionally) methionine at a concentration of about 0 to about 100 mM, eg, 100 mM; and (f) a Histidine (at a concentration of about 10 to about 20 mM) or a Tris buffer (at a concentration of about 20 mM), such that the pH of the formulation is from about 5 to 7.5, e.g. , 5.5, 5.8-6.1, 6, 6.1, 6.5 or 7.

In another aspect, the invention relates to a method of treating or preventing in a subject (e.g., a human subject) a disorder associated with aAPT, e.g., inflammatory or autoimmune disorders, including, but not limited to, rheumatoid arthritis. (AR) (for example, moderate to severe rheumatoid arthritis), arthritic conditions (for example, arthritis psoriatic, juvenile idiopathic arthritis (JIA) polyarticular, ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease and / or multiple sclerosis. The method includes administering to a subject, for example, a human patient, a pharmaceutical composition that includes a TNF-binding UADS formulation, as described herein, for example, a formulation containing a binding UADS molecule. to TNF, alone or in combination with any of the combination therapies described herein, in such an amount that one or more of the symptoms of the disorder associated with TNFct is reduced.

In another aspect, the invention features a kit or article of manufacture that includes a device, a syringe or a vial containing the formulations described herein. The kit or article may optionally include instructions for its use. In some embodiments, the syringe or vial is made of glass, plastic or a polymeric material, such as cyclic olefin polymer or copolymer. In other embodiments, the formulation may be presented in an injectable device (e.g., an injectable syringe, e.g., a pre-filled injectable syringe). The syringe can be adapted for individual administration, for example, as a simple vial system that includes an auto-injector (e.g., a pen injector device) and / or instructions for its use. In one embodiment, the injectable device is a pre-filled pen or other suitable self-injecting device, optionally with instructions for its use and administration.

In some embodiments, the subject is provided with the kit or article of manufacture (e.g., the pen or syringe previously loaded with a single or multiple dose unit), e.g., a patient or a health care provider, already packaged with instructions for administration (eg, self-administration) pro injection. { for example, subcutaneous, intravascular, intramuscular or intraperitoneal).

In other embodiments, the invention features a device for nasal, transdermal and intravenous administration of the described formulations provided herein. For example, a transdermal patch is provided for the administration of the formulations described herein. In still other cases, an intravenous bag is provided for the administration of the formulations described herein. In some embodiments, the intravenous bag is provided with normal saline or 5% dextrose.

In another aspect, the invention features a method for teaching the patient (e.g., a human patient), in need of said UADS molecule, e.g., a nanobody molecule of TNFa, how to administer a formulation described herein. The method includes: (i) providing the patient with at least one unit dose of a formulation of the UADS molecule described herein; and (ii) teaching the patient how to self-administer at least one unit dose, e.g., by injection (e.g., subcutaneous, intravascular, intramuscular or intraperitoneal). In one embodiment, the patient suffers from a disorder associated with TNFa, for example, inflammatory or autoimmune disorders as described herein.

In another aspect, the invention features a method for teaching a recipient about the administration of a formulation of a nanobody molecule of TNFα described herein. The procedure includes teaching the recipient (for example, a patient end user, doctor, retail pharmacy or wholesaler, distributor or pharmacy department of a hospital, nursing home clinic or OMH (Organization for Health Maintenance)) as it should be. administer the formulation to the patient.

In another aspect, there is provided a method of distributing a formulation of a UADS molecule, for example, a nanobody molecule of TNFα, described herein. The method includes providing a recipient (e.g., an end user, patient, physician, retail pharmacy or wholesaler, distributor or pharmacy department of a hospital, clinic of a nursing home or OMH) with a container containing unit dosages of the UADS molecule, for example, a nanobody molecule of TNFa, sufficient to treat a patient for at least 6, 12, 24 or 36 months.

In another aspect, the invention presents a method or method for evaluating the quality of a package or package lot. { by example, to determine if it has expired) of a formulation described herein containing a UADS molecule, for example, a nanobody molecule of TNFa. The procedure includes evaluating if the container has expired. The expiration date is at least 6, 12, 24, 36 or 48 months, for example, greater than 24 or 36 months, from a preselected event, such as manufacturing, testing or packaging. In some embodiments, as a result of the analysis, a decision is made or a step is made, for example, the UADS molecule in the package is used or rejected, sorted, selected, distributed or retained, mapped, it moves to a new location, it is distributed to the trade, it is sold or offered for sale, it is removed from the trade or it is no longer offered for sale, depending on whether the product has expired.

In another aspect, the invention features a method for storing, distributing or using a formulation of a UASD molecule, for example, a TNF nanobody molecule, described herein. The method includes: preserving the formulation for a period at a certain temperature, for example, less than 25 ° C, for example, below the freezing point or below 15 ° C, 10 ° C or 4 ° C. In some embodiments, the method further includes providing the formulation to a recipient, e.g., to an end user, e.g., a patient or healthcare provider, to store in similar or different conditions (e.g., a temperature greater than the of the first storage period). The formulation may be a liquid, lyophilized or reconstituted formulation.

In another aspect, the invention features a method for analyzing a product or process, for example, a manufacturing process. The method includes providing a formulation of a UADS molecule, for example, a nanobody molecule of TNF, as described herein, and evaluating a parameter of the formulation, such as color (e.g., colorless to slightly yellow). or from colorless to yellow), transparency (for example, from light to slightly opalescent or clear to opalescent) or viscosity (for example, between about 1 to 5 cP when measured at room temperature, such as at 20 ° C- 30 ° C, for example, 25 ° C), quantity of one or more species of AP, BPM, acid and / or basic, as described in this document. The evaluation may include an assessment of one or more parameters. Optionally, it is determined if the parameter complies with a certain preselected criterion, for example, it is determined if the preselected criterion is present, or is present in a pre-selected interval, analyzing in this way the procedure.

In one embodiment, the evaluation of the method includes measuring the stability of the formulation of the UADS molecule. The stability of the antibody formulation can be measured, for example, by the formation of aggregates, which is tested, for example, by high pressure liquid chromatography by size exclusion (HPLC-SE), by color, transparency or viscosity as described herein. A formulation can be determined to be stable and therefore acceptable for further processing or distribution, if the change in a test parameter is less than about 10%, 5%, 3%, 2%, 1%, 0.5% , 0.05% or 0.005% or less, during a pre-established period of time and optionally at a certain temperature.

In one embodiment, the method further includes comparing the determined value with a reference value, to thereby analyze the manufacturing process.

In one embodiment, the method further includes preserving the manufacturing process based, at least in part, on the analysis. In one embodiment, the method further includes modifying the manufacturing process based on the analysis.

In another embodiment the method includes evaluating a method, e.g., a method of making a formulation of a UADS molecule, e.g., a TNF nanobody molecule, manufactured by a selected method, including performing a procedure determination. based on a procedure or analysis described in this document. In one embodiment, the method further includes preserving or modifying the manufacturing process based, at least in part, on the process or analysis. Therefore, in another embodiment, the part of performing the evaluation does not follow the procedure or analysis described herein. document but only based on results obtained through a procedure or analysis described in this document.

In another embodiment the method includes comparing two or more preparations in a batch to batch variation monitoring or control procedure or comparing a preparation with a reference standard.

In yet another embodiment, the method may additionally include making a decision, for example, classifying, selecting, accepting or rejecting, distributing or retaining, processing a pharmaceutical product, shipping, moving to another location, formulating, labeling, packaging, distributing to the merchant , sell or offer for sale the preparation, based, at least in part, on the determination.

In another aspect, the invention presents a method for evaluating the quality of a formulation of a UADS molecule, for example, a nanobody molecule of TNF as described herein, for example, in a quality control analysis or distribution specification. The method includes providing an evaluation of a formulation of a UADS molecule for a parameter, such as color (for example, colorless to slightly yellow or colorless to yellow), transparency (for example, from transparent to slightly opalescent or transparent). to opalescent) or viscosity (e.g., between about 1 to 5 cP when measured at room temperature, such as 20 ° C to 30 ° C, eg, 25 ° C). The evaluation may include assessing one or more of the above parameters. The procedure also it optionally includes determining whether the solution parameter meets a preselected criterion, for example, if the preselected criterion is present or is present in a pre-selected interval. If the parameter of the observed solution is within a preselected range of values or meets the pre-selected standards criteria, then the preparation is selected, such as for packaging, use, sale, distribution to trade, rejection, etc.

In another aspect, the invention presents a method for complying with a regulatory requirement, for example, a requirement of a regulatory agency subsequently approved, for example, the FDA (drug and food administration). The method includes providing an evaluation of an antibody formulation for a parameter, as described herein. The subsequently approved requirement may include a measurement of one or more of the above parameters. The method also optionally includes determining whether the parameter of the observed solution meets a preselected criterion or whether the parameter is in a preselected range; optionally, register the value or result of analysis or communicate with the agency, for example, transmitting the value or result to the regulatory agency.

In another aspect, the invention features a method for manufacturing a batch of a formulation of a UADS molecule, for example, a nanobody molecule of TNF, having a preselected property, for example, that meets a distribution specification, a labeling requirement or a summary requirement, for example, a property described in this document. The method includes providing an assay formulation; analyzing the test formulation, according to a procedure described herein; determine if the test formulation satisfies a preselected criterion; for example, having a preselected relationship with a reference value, for example, one or more reference values described herein and selecting the test antibody preparation for manufacturing a batch of product.

In another aspect, the invention features multiple batches of a formulation of a UADS molecule, for example, a nanobody molecule of TNF, in which, for each batch, one or more parameters. { for example, a solution value or parameter determined by a method described herein) varies less than a preselected range from a preselected desired reference value or criterion, e.g., a range or criterion described herein. In some embodiments, for one or more batches of formulation, one or more parameters are determined and as a result of the determination one or more batches are determined. Some embodiments include comparing the results of the determination with a preselected value or criterion, for example, a reference standard. Other embodiments include adjusting the dose of the batch to be administered, for example, based on the result of the determination of the value or parameter.

In another aspect, the invention features a method of one or more of: providing a report to a reporting entity, evaluating a sample of a formulation of a UADS molecule, eg, a nanobody molecule of TNF, in compliance with a reference standard, for example, a requirement of the FDA, requesting indications to the other party that a preparation of the UADS molecule meets some predefined requirement or present information to the other party about a preparation of a UADS molecule. Exemplary entities or other receiving parties include a government, for example, the federal government of the United States, for example, a government agency, for example, the FDA. The method includes one or more (or all) of the following steps for manufacturing and / or testing an aqueous formulation of the UADS molecule in a first country, eg, the United States.; send at least one aliquot of the sample outside the first country, for example, send it outside the United States, to a second country; preparing, or receiving, a report that includes data on the structure of the preparation of the UADS molecule, for example, data related to a structure and / or chain described herein, for example, data generated by one or more of the procedures described in this document and provide said report to a receiving entity.

In one embodiment, the reporting entity can determine whether a predetermined requirement or reference value matches the data and optionally, a response is received from the receiving entity. reports, for example, by a distributor or vendor manufacturer of a UADS molecule formulation. In one embodiment, after receiving approval from the reporting entity, the preparation of a UADS molecule formulation is selected, packaged and marketed.

In another aspect, the invention features a method for evaluating a formulation of a UADS molecule. The method includes receiving data regarding the presence or level of a UADS molecule, for example, in which the data was prepared by one or more methods described herein; provide a record including said data and optionally include an identifier for a batch of a UADS molecule; submit such registration to an authority, for example, a government agency, for example, the FDA; optionally, receive a communication from said authority; optionally, decide the distribution or commercialization of the UADS molecule lot based on the communication from the authority. In one embodiment, the method further includes distributing the sample.

All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Although in the practical embodiment or assay of the present invention, methods and materials similar or equivalent to those described herein can be used, suitable procedures and materials are described below. In addition, the materials, procedures and examples are illustrative only and are not intended to be limiting.

From the detailed description, drawings and claims, other features and advantages of the present invention will be apparent.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 depicts results of the biological activity of a lyophilized formulation of 106 U / mg of TNF-binding nanobody (ATN-103) stored as a dry powder preparation (PS) for up to six months. The formulation was stored at the indicated temperatures.

Fig. 2 depicts results of binding activity to Human Serum Albumin (ASH) of a lyophilized formulation of TNF-binding nanobody (ATN-103). The results are shown as a percentage (%) of the reference pattern of the TNF-binding nanobody.

Fig. 3 depicts HPLC results by size exclusion (HPLC-SE) in% of high molecular weight species (APM) for the lyophilized formulation.

FIG. 4 depicts capillary electrophoresis results in SDS (CE-SDS) in% of TNF-binding nanobody for the lyophilized formulation.

FIG. 5 depicts HPLC-SE results for% APM species for the formulation subject to control lyophilization and solidity cycles.

FIG. 6 depicts results of the biological activity of 106 U / mg of the TNF binding nanobody after storage for up to six months of a liquid formulation at high concentration.

Fig. 7 depicts results of binding activity to Albumin of Human Serum (ASH) (percentage of Reference Standard of nanobody of union to TNF) of a liquid formulation at high concentration stored up to six months at the indicated temperatures.

FIG. 8 depicts HPLC-SE results for the% APM species of a liquid formulation at high concentration after storage for up to six months at the indicated temperatures.

Fig. 9 represents HPLC-SE results for the% of BPM species of a liquid formulation at high concentration after storage for up to six months at the indicated temperatures.

FIG. 10 depicts CE-SDS results for% ATN-103 of a liquid formulation at high concentration after storage for up to six months at the indicated temperatures.

FIG. 11 depicts HPLC-SE results for the% APM species of a high-concentration liquid formulation in a pre-filled syringe.

FIG. 12 depicts HPLC-SE results for% of BPM species of a high-concentration liquid formulation in a pre-filled syringe.

Fig. 13 represents results of% acid species by HPLC-CEX of a high-concentration liquid formulation in a pre-filled syringe.

Fig. 14 represents results of the% of basic species by HPLC-CEX of a liquid formulation at high concentration in a syringe previously loaded.

Fig. 15 represents results of the% of APM species, by HPLC-SE, of liquid formulations at high concentration - Other Formulations (identification of other stabilizing and destabilizing excipients) Fig. 16 represents results of the% of APM species, by HPLC-SE, of a liquid formulation at high concentration of the TNF-binding nanobody.

Fig. 17 represents results of the% of BPM species, by HPLC-SE, of a liquid formulation at high concentration of the TNF binding nanobody.

Fig. 18 represents results of% acid species, by HPLC-CEX, of a liquid formulation at high concentration of the TNF-binding nanobody.

Fig. 19 represents results of the% of basic species, by HPLC-CEX, of a liquid formulation at high concentration of the TNF-binding nanobody.

FIG. 20 depicts results of the% APM species, by HPLC-SE, of a high concentration liquid formulation of the TNF binding nanobody after 10 freeze-thaw cycles.

Fig. 21 represents results of the% of BPM species, by HPLC-SE, of a liquid formulation at high concentration of the TNF-binding nanobody after 10 freeze-thaw cycles.

Fig. 22 represents the Turbidity (Absorbance at 455 nm) of the liquid formulation at high concentration of the TNF-binding nanobody after 10 freeze-thaw cycles.

Fig. 23 represents the Concentration (by UV absorbance at 280 nm) of the liquid formulation at high concentration of the TNF-binding nanobody after 10 freeze-thaw cycles.

Fig. 24 represents the% of APM species, by HPLC-SE, of the liquid formulation at high concentration of the TNF-binding nanobody: after short-term thermal stress possibly found in manufacturing processes.

Fig. 25 represents results of% APM species, by HPLC-SE, of the liquid formulation at low concentration as a function of pH and formulation (40 ° C).

Fig. 26 represents results of the% of BPM species, by HPLC-SE, of the liquid formulation at low concentration as a function of pH and formulation (40 ° C).

Fig. 27 represents results of the% APM species, by HPLC-SE, of the liquid formulation at low concentration as a function of pH and formulation (4 ° C).

Fig. 28 represents results of the% of APM species, by HPLC-SE, of the liquid formulation at low concentration as a function of pH and formulation after stirring.

Fig. 29 represents a schematic diagram of the predicted ATN-103 structure.

Fig. 30 represents the amino acid sequence of the polypeptide chain of ATN-103 (SEQ ID NO: 1).

Fig. 31 corresponds to bar graphs representing the% of APM species, detected by HPLC-SE, of the formulations indicated containing approximately 100 mg / ml of ATN-103 (HST, HSGT, HSGMT, HSorb and control) stored in the indicated conditions.

Fig. 32 corresponds to bar graphs representing the% of BPM species, detected by HPLC-SE, of the formulations indicated containing approximately 100 mg / ml of ATN-103 (HST, HSGT, HSGMT, HSorb and control) stored under the indicated conditions. At the initial time point or after two weeks at 4 ° C, no BPM species were detected.

DETAILED DESCRIPTION OF THE INVENTION It has been identified that stable formulations that include a UADS molecule, eg, a nanobody molecule (e.g., a TNF-binding nanobody molecule), are suitable for the preservation of high and low concentrations of the UADS molecule. (a "formulation"). The UADS molecule that is formulated is preferably essentially pure and desirably essentially homogeneous (ie, free of contaminating proteins, etc.). "Essentially pure" protein means a composition comprising at least about 90% by weight of the protein, based on the total weight of the composition, preferably at least about 95% by weight. "Essentially homogeneous" protein means a composition comprising at least about 99% by weight of protein, based on the weight of the composition.

The integrity of the UADS molecule in the formulation is generally retained by following a long-term storage as a liquid or lyophilized product under various conditions. For example, the integrity of the UADS molecule is properly conserved after exposure over a wide range of storage temperatures (e.g., -80 ° C to 40 ° C), shear (e.g., agitation), and interfacial stress (freeze-thaw cycles).

Additionally, for lyophilized material, the integrity of the UADS molecule is adequately preserved during the reconstitution process. In addition, the integrity of the UADS molecule is sufficiently conserved for use as a medicament as demonstrated by relatively low accumulations of BPM species and AP species, in vitro bioactivity, in vitro binding activity after long-term storage (e.g. up to 12 months) at various temperatures (for example, -80 ° C to 40 ° C).

In order that the present invention can be better understood, some terms are defined first. Additional definitions are set forth throughout the detailed description.

As used in this document, articles "one" and "one" refers to one or more than one (for example, at least one) of the grammatical object of the article.

The term "or" as used herein, refers to and is used interchangeably with the term "and / or", unless the context clearly dictates otherwise.

The terms "proteins" and "polypeptides" are used interchangeably herein.

"Around" and "approximately" will generally mean an acceptable degree of error of the measured quantity given the nature or accuracy of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10% and more typically, within 5% of a given value or range of values.

A "stable" formulation of a UADS molecule shows little or no symptoms of any or more aggregation, fragmentation, deamidation, oxidation or change in biological activity over a prolonged period of time, eg, 6, 12 months, 24 months , 36 months or more. For example, in one embodiment, less than 10% of the UADS molecule is added, fragmented or oxidized. Aggregation, precipitation and / or denaturation can be evaluated by known methods, such as color visual examination and / or transparency or by UV light scattering or size exclusion chromatography. The ability of the protein to preserve its biological activity can be assessed by detecting and quantifying chemically modified forms of the antibody. The size modification (for example, trimming), which can be evaluated using size exclusion chromatography and / or SDS-PAGE, for example. Other types of chemical modification include charge modification (e.g., that occurs as a result of deamidation), which can be evaluated by ion exchange chromatography, for example.

A UADS molecule "retains its biological activity" in a pharmaceutical formulation, if the biological activity of the molecule in a determined time is within about 50% or more of the biological activity shown at the time of the pharmaceutical formulation prepared as determined in an antigen-binding assay, for example.

A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein formulation in a diluent such as the protein that is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (eg, parenteral or peripheral administration) to a patient to be treated with the protein of interest and in certain embodiments of the invention may be one that is suitable for subcutaneous administration.

"Isotonic" or "so-osmotic" means that the formulation of interest has similar or essentially the same osmotic pressure as human blood. Isotonic or osmotic formulations will generally have an osmotic pressure of about 250 to 350 mOsm. The sotonicity can be measured using a vapor pressure or cryoscopic osmometer, for example.

A "tonicity adjusting agent" refers to a compound that makes the formulation substantially sotonic or osmotic with human blood. Exemplary tonicity adjusting agents are: sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, or arginine hydrochloride. Typically, the tonicity adjusting agents are added in an amount such that the overall formulation exerts an osmotic resistance similar to that of human blood. By For example, human blood contains approximately 300 mM solutes. Typically, pharmaceutical products direct a total molarity of 300 mM. This corresponds to an osmotic pressure of approximately 300 to 310 mOsm, with a typical range of 250 mOsm to 350 mOsm. The amount of tonicity adjusting agent required can be estimated initially by calculation. The contribution with respect to the total molarity can be estimated from the molecular weight of a molecule of excipient and known properties of the molecule, for example, if the molecule dissociates into two ionic species or the molecule is non-ionic (does not dissociate) . Additionally, it is necessary to understand the osmotic contribution of the specific protein molecule as a function of protein concentration. This parameter can be determined experimentally.

For example, starting with a formulation (uncorrected tonicity) of 10 mM histidine, 5% sucrose, 0.01% polysorbate 80, with a concentration of anti-TNF nanobody protein of 100 mg / ml, as a first stage , the estimated molarity of the starting formulation can be calculated as follows: 10 mM histidine = 10 mM Sucrose 5% corresponds approximately to 146 mM 5% = 5 g / 100 ml = 50 g / l? (50 g / l) / (342.3 g / mol) = 0.146 mol / l = 146 mM Polysorbate 80 at 0.01% essentially exerts a zero molarity and can be discarded. The 100 mg / ml protein was determined through the experimentation so that 100 mg / ml of anti-TNF nanobody protein exerts an osmotic pressure corresponding to approximately 48 mM.

Therefore, adding all the contributions for the molarity in the initial formulation: 10 mM + 146 mM + 48 mM = 204 mM.

If the target molarity is 310 mM, then the corresponding quantity molarity to compose the rest of the target is: 310 mM - 204 mM = 106 mM Therefore, the recommended amount of tonicity adjusting agent is 106 mM of a non-ionic tonicity adjusting agent or 53 mM of an ionic tonicity adjusting agent that completely dissociates into two ionic species.

After determining the initial calculation of the tonicity adjusting agent, it is recommended to test the formulation experimentally. Therefore, in the provided example, 100 mM glycine was added to the initial formulation. (The recommended 106 mM was rounded down to 100 mM for simplicity). The expected osmolarity would be: 10 mM histidine + 146 mM sucrose + 48 mM protein + 100 mM glycine = 304 mM The value of the experimental osmotic pressure of the formulation is = 305 mOsm.

A "lyoprotectant" is a molecule that, when combined with a protein of interest, prevents or significantly reduces the chemical and / or physical instability of the protein after lyophilization and subsequent storage. Exemplary lyoprotectants include sugars such as sucrose, sorbitol or trehalose; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher sugar alcohols, for example, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; pluronic and combinations thereof. Typically, the lyoprotectant is a non-reducing sugar, such as trehalose or sucrose. The lyoprotectant is added to the pre-lyophilized formulation in a "lyoprotective amount" which means that, after lyophilization of the protein in the presence of the lyoprotectant amount of the lyoprotectant, the protein essentially retains its physical and chemical stability and integrity after of lyophilization and storage.

A "stabilizer" refers to a molecule that, when combined with a protein of interest (e.g., the UADS molecule), substantially prevents or reduces chemical and / or physical instability of the protein of interest in lyophilized, reconstituted, liquid form or storage. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine and arginine hydrochloride.

The "diluent" of interest in the present document is one that is pharmaceutically acceptable (innocuous and non-toxic for administration in a human) and is useful for the preparation of a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (ABPI), a buffered solution with pH (eg, phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution.

A "preservative" is a compound that can be added to the diluent to essentially reduce the bacterial action in the reconstituted formulation thereby facilitating the production of a multi-use reconstituted formulation, for example. Examples of possible preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethyl ammonium chlorides in which the alkyl groups are long chain compounds) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative herein is benzyl alcohol.

A "bulking agent" is a compound that adds bulk to the lyophilized mixture and contributes to the physical structure of the lyophilisate (eg, facilitates the production of an essentially uniform lyophilizate that retains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol and sorbitol.

The methods and compositions of the present invention include polypeptides and nucleic acids having the specified sequences or sequences substantially identical or similar thereto, eg, sequences with an identity of at least 85%, 90% and 95% or greater with the specified sequence. In the context of an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of amino acid residues aligned in a second amino acid sequence so that the first and second amino acid sequence may have a common structural domain and / or a common functional activity. For example, the amino acid sequences containing a common structural domain have at least approximately an identity of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with respect to a reference sequence. In other embodiments, the amino acid sequence may contain one or more amino acid insertions, deletions or substitutions (e.g., conservative substitutions) to arrive at a percent identity of at least about 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with respect to the reference sequence.

In the context of nucleotide sequence, the term "substantially identical" as used herein refers to a first nucleic acid sequence that contains a sufficient number or at least nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having a common functional activity or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, the nucleotide sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with respect to a reference sequence.

Also included as polypeptides of the present invention are fragments, derivatives, analogs or variants of the above polypeptides and any combination thereof. The terms "fragment", "variant", "derivative" and "analogue" when referring to proteins of the present invention include any of the polypeptides that retain at least one of the functional properties of the corresponding natural antibody or polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments described elsewhere herein. Variants of the polypeptides of the present invention include fragments as described above and also polypeptides with modified amino acid sequences due to substitutions, deletions or amino acid insertions. The variants can be of natural origin or of non-natural origin. Variants of natural origin can be produced using mutagenesis techniques known in the art. Variant polypeptides can understand conservative or non-conservative substitutions, deletions or additions of amino acids. The derivatives of the fragments of the present invention are polypeptides that have been modified to show additional characteristics not found in the natural polypeptide. Examples include fusion proteins. Variant polypeptides are also referred to herein as "polypeptide analogs." As used herein, a "derivative" of a polypeptide refers to a subject polypeptide having one or more chemically derivatized moieties by reaction of a functional side group. Also included as "derivatives" are polypeptides that contain one or more naturally occurring amino acid derivatives of the twenty conventional amino acids. For example, 4-hydroxyproline can substitute proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; Homoserine can replace serine and ornithine can replace lysine.

The term "functional variant" refers to polypeptides having an amino acid sequence substantially identical to the sequence of natural origin or encoded by a substantially identical nucleotide sequence and may have one or more naturally occurring sequence activities.

The homology or sequence identity calculations between the sequences (terms in this document are used interchangeably) are performed in the following manner.

To determine the percent identity of two amino acid sequences, or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of a first and a second amino acid sequence or of nucleic acid for optimal alignment and non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides in the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as in the corresponding position in the second sequence then the molecules are identical in that position (as used herein amino acid or as used herein) "identity" of amino acids or nucleic acid is equivalent to "homology" of amino acids or nucleic acid).

The percentage of identity between two sequences is a function of the number of identical positions that the sequences share, taking into account the number of holes and the length of each gap that needs to be introduced for the optimal alignment of the two sequences.

The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch ((1970) J. Mol. Biol. 48: 444-453) which has been incorporated into the GAP program in the package GCG computer (available at http://www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1 , 2, 3, 4, 5 or 6. In another preferred additional embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com) using an NWSgapdna.CMP matrix and a hole weight of 40, 50, 60, 70 or 80 and a length weight of 1 2, 3, 4, 5 or 6. A series of particularly preferred parameters (and one that would be used unless specified otherwise) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4 and a gap penalty of the gap reading frame of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989), CABIOS 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a table of weight remains PAM120, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a "problem sequence" to perform a search against public databases with respect, for example, to identifying other members of the family or related sequences. Said searches can be performed using the NBLAST and XBLAST (version 2.0) programs of AltschuI, et al (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 00, word length = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention (SEQ ID NO 1). Searches for BLAST proteins can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to a protein molecule of a protein (SEQ ID NO 1) of the invention. To obtain gap alignments for comparison purposes, Gapped BLAST can be used as described in AltschuI et al, (1997) Nucleic Acids Res. 25: 3389-3402. When BLAST and Gapped BLAST programs are used, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acid side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), branched beta side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

Various aspects of the invention are described in greater detail below.

Simple Domain Antigen Binding Molecules (UADS) Single domain antigen binding molecules (UADS) include molecules whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, naturally occurring light chain-deprived binding molecules, single domains derived from conventional 4-chain antibodies, engineered domains, and frameworks from simple domains other than those derived from antibodies The UADS molecules may be any of the art or any future single domain molecule. UADS molecules can be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit and bovine. East The term also includes single domain antibody molecules of natural origin from species other than Camelids and sharks.

In one aspect of the invention, the UADS molecule can be derived from a variable region of the immunoglobulin found in fish, such as, for example, the one derived from the immunoglobulin isotype known as the new antigen receptor (NAR) found in the serum of shark. Methods for producing single domain molecules derived from a variable region of NAR ("IgNARs") are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14: 2901-2909.

According to another aspect of the present invention, a UADS molecule is a single domain antigen-binding molecule of natural origin which is known as heavy chain devoid of light chains. Such single domain molecules are disclosed in WO 9404678 and Hamers-Casterman, C. et al (1993) Nature 363: 446-448, for example. To clarify, this variable domain derived from a heavy chain molecule devoid of naturally occurring light chain is referred to herein as a VHH or nanobody to differentiate it from the conventional four chain immunoglobulin VH. Said VHH molecule may come from Camelid species, for example camel, llama, dromedary, alpaca and guanaco. Other species together with Camelids can produce heavy chain molecules naturally devoid of light chain; such VHH are within the scope of the invention.

The UADS molecules can be recombinant, CDR-grafted, humanized, camelized, deimmunized and / or generated in vitro (eg selected by phage display) as described in more detail below.

The term "antigen binding" is intended to include that portion of a polypeptide, for example, a single domain molecule described herein, comprising determinants that form an interface that binds to a target antigen or an epitope thereof. With respect to proteins (or protein mimetics), the specific antigen-binding site includes one or more loops (of at least four amino acids or amino acid mimetics) that form an interface that binds to the target antigen. Typically, the antigen-binding site of the polypeptide, eg, the single-domain antibody molecule, includes at least one or two CDRs, or more typically at least three, four, five or six CDRs.

The term "immunoglobulin variable domain" is frequently understood in the art to be identical or substantially identical to a VL domain or a VH domain of human or animal origin. It should be recognized that the variable domain of immunoglobulin may have evolved in certain species, for example, sharks and llama to differentiate into amino acid sequences of humans or mammals of VL or VH. However, these domains are primarily involved in antigen binding. The expression "variable domain of "immunoglobulin" typically includes at least one or two CDRs or more typically at least three CDRs.

A "constant immunoglobulin domain" or "constant region" is intended to include an immunoglobulin domain that is identical or substantially identical to a CL, CH1, CH2, CH3 or CH4 domain, of human or animal origin. See, for example, Charles A Hasemann and J. Donald Capra, Immunoglobulins: Structure and Function, in William E. Paul, ed., Fundamental Immunology, second edition, 209, 210-218 (1989). The term "Fe Region" refers to the Fe part of the immunoglobulin constant domain that includes immunoglobulin CH2 and CH3 domains or immunoglobulin domains substantially similar to these.

In some embodiments, the UADS molecule is a monovalent or multispecific molecule (for example as a bivalent, trivalent or tetravalent molecule). In other embodiments, the UADS molecule is a monospecific, bispecific, trispecific or tetra-specific molecule. If a molecule is "monospecific" or "multispecific", for example, "bispecific", it refers to the number of different epitopes with which a binding polypeptide reacts. The multispecific molecules may be specific for different epitopes of a target polypeptide described herein or may be specific for a target polypeptide as well as for a heterologous epitope such as heterologous polypeptide or solid support material.

As used herein the term "valence" refers to the number of possible binding domains, for example, domains of antigen binding, present in a UADS molecule. Each binding domain binds specifically to an epitope. When a UADS molecule comprises more than one binding domain, each binding domain can bind specifically to the same epitope, to an antibody with two binding domains, termed "monospecific bivalent", or to different epitopes for a UADS molecule with two domains of union, called "bispecific bivalent". A UADS molecule can also be bispecific and bivalent for each specificity (termed "bispecific tetravalent molecules"). Bivalent and bispecific molecules and methods for preparing them are described, for example, in U.S. Patent Nos. 5,731, 168; 5,807,706; 5,821, 333; and in U.S. Application Publication No. 2003/020734 and 2002/0155537, the complete descriptions of which are hereby incorporated into this document. Bispecific tetravalent molecules and methods for preparing them are described, for example, in WO 02/096948 and WO 00/44788, the descriptions of which are incorporated by reference herein. See, generally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al, J. Immunol. 147: 60-69 (1991); U.S. Patent No. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601, 819; Kostelny et al, J. Immunol. 148: 1547-1553 (1992).

In some embodiments, the UADS molecule is a single chain fusion polypeptide comprising one or more single domain molecules (e.g., nanobodies), devoid of a variable domain complementary or a constant region of immunoglobulin, for example, Fe, which binds to one or more target antigens. An exemplary target antigen recognized by the antigen-binding polypeptides includes tumor necrosis factor a (TNF a). In some embodiments, the single-domain antigen-binding molecule binds to a serum protein, for example, a human serum protein selected from one or more of serum albumin (human serum albumin (HSA)) or transferin.

TNFa It is known in the art that tumor necrosis alpha factor is associated with inflammatory diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. Both TNFa and the receptors (CD120a and CD120b) have been studied in great detail. TNFa in its bioactive form is a trimer. Several strategies have been developed to antagonize the action of TNFα using anti-TNFα antibodies and are currently available on the market such as Remicade® and Humira®. Antibody molecules against TNFa are known. Numerous examples of single-domain antigen binding molecules to TNFa (e.g., nanobodies) are disclosed in documents WO / 2004/041862, WO 2004/041865, WO 2006/122786 the complete contents of which are incorporated herein by reference in their entirety. Additional examples of antigen-binding molecules of simple domain are disclosed in the documents US 2006/286066, US 2008/0260757, WO 06/003388, US 05/0271663, US 06/0106203, the total contents of which are incorporated herein by reference in their entirety. In other embodiments, mono, bi, tri and other multispecific single domain antibodies against TNFa and a serum protein, eg, ASH, are disclosed in these references.

In specific embodiments, the TNFα binding nanobody molecule comprises one or more of the nanobodies described in WO2006 / 122786. For example, the TNFα binding nanobody molecule can be a monovalent, bivalent, trivalent TNFα binding nanobody molecule disclosed in WO2006 / 122786. Exemplary TNFα binding nanobodies include, but are not limited to, TNF1, TNF2 , TNF3, humanized forms thereof (for example, TNF29, TNF30, TNF31, TNF32, TNF33). Additional examples of monovalent TNF [alpha] binding nanobodies are disclosed in Table 8 of WO2006 / 122786. Exemplary bivalent TNFα-binding nanobody molecules include, but are not limited to, TNF55 and TNF56, which comprise two TNF30 nanobodies linked by peptide linkage to form a single fusion polypeptide (disclosed in WO2006 / 122786). Additional examples of bivalent TNFα-binding nanobody molecules are described in Table 19 of WO2006 / 122786 as TNF4, TNF5, TNF6, TNF7 and TNF8).

In other embodiments, the ASH-binding nano-body molecule comprises one or more of the nanobodies described in WO2006 / 122786. For example, the ASH-binding nanobody molecule can be a monovalent, bivalent, trivalent ASH-binding nanobody molecule described in WO2006 / 122786. In other embodiments, the ASH-binding nanobody molecule can be a monospecific or multispecific molecule having at least one of the binding specificities linked to ASH. Exemplary TNFa binding nanobodies include, but are not limited to, ALB1, humanized forms thereof (eg, ALB6, ALB7, ALB8, ALB9, ALB10), disclosed in WO 06/122786.

In other embodiments, two or more of the single domain molecules of the nanobody molecules are fused, with or without a linker group, such as a polypeptide or genetic fusion. The linker group can be any linkage group apparent to those skilled in the art. For example, the linker group can be a biocompatible polymer with a length of 1 to 100 atoms. In one embodiment, the linking group includes or consists of polyglycine, poly-serine, polylysine, polyglutamate, polyisoleucine or polyarginine moieties or a combination thereof. For example, the polyglycine or polyserin linkers may include at least five, seven, eight, nine, ten, twelve, fifteen, twenty, thirty, thirty-five and forty glycine and serine residues. The exemplary linkers that can be used include Gly-Ser repeats, for example, repetitions of (GlyVSer (SEQ ID NO 8) of one, two, three, four, five, six, seven or more repeats In embodiments, the bond has the following sequences: (Gly) 4-Ser- (Gly) 3-Ser ( SEQ ID NO 9) or ((Gly) 4-Ser) n (SEQ ID NO 10), where n is 4, 5 or 6.

In an exemplary embodiment, an antigen-binding polypeptide composed of a single-chain polypeptide fusion of two single-domain antibody molecules (e.g., two camelid variable regions) that bind to a target antigen, e.g. alpha factor of tumor necrosis (TNFa) and a single-domain antibody molecule (e.g., a variable region of camelid) that binds to a serum protein, e.g., ASH, referred to herein as "ATN-103" was shown to be linked to protein A or a functional variant thereof. ATN-103 is a bispecific trivalent humanized fusion protein, of inhibition of TNFa. The antigen for this protein is tumor necrosis factor-alpha (TNF). Figure 29 provides a schematic representation of the predicted structure of ATN-103. This fusion protein is derived from camelids and has a high degree of sequence and structural homology with respect to the VH domains of human immunoglobulin. Its simple polypeptide chain is composed of two TNFa binding domains and one human serum albumin (ASH), with two G-S linkers of nine amino acids that connect the domains. A detailed description of ATN-03 is provided in WO 06/122786.

The complete amino acid sequence of the polypeptide chain ATN-103 predicted from the DNA sequence of the corresponding expression vector is shown in Figure 30 (the residues are numbered starting with the NH 2 terminus as residue number 1 of SEQ ID NO 1 ). The last amino acid residue encoded by the DNA sequence is S363 and constitutes the COOH end of the protein. The molecular mass predicted for ATN-103 bound by disulfide bridges (without post-translational modifications) is 38434.7 Da. ATN-103 does not contain consensus sequences of N-linked glycosylation. The molecular mass observed for the predominant isoform by quadrupole time-of-flight mass spectrometry with nanoelectrospray ionization corresponds to 38433.9 Da, which confirms the absence of post-translational modifications.

In Figure 30, the regions determining complementarity (CDR) are underlined. The predicted intramolecular disulfide bonds are illustrated by connections of the cysteine residues involved. The TNF binding domains are shown in bold type and the ASH binding domain is shown in italics and bold. The linkers of amino acids that connect these binding domains are in italics. The signal peptide ("19 MGW ... VHS" 1) is also shown for the polypeptide chain.

Preparation of UADS Molecules The UADS molecules may comprise one or more molecules of single domains (e.g., nanobodies) that are recombinants, grafted to CDR, humanized, camelized, deimmunized and / or generated in vitro (for example, selected by phage display). Techniques for generating antibodies and UADS molecules and modifying them recombinantly are known in the art and are described in detail below.

Numerous methods known to those skilled in the art are available to obtain antibodies. For example, monoclonal antibodies can be produced by generating hybridomas according to known procedures. Hybridomas formed in this way are then screened using conventional methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis (BIACORE ™), to identify one or more hybridomas that produce a nanobody that binds specifically to a specific antigen. Any form of the specified antigen can be used as the immunogen, for example, recombinant antigen, forms of natural origin, any variant or fragment thereof as well as antigenic peptides thereof.

An exemplary method for preparing antibodies and UADS molecules includes screening protein expression libraries, e.g., phage display libraries or ribosomes. In Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228: 1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809 describe phage displays.

In addition to the use of presentation libraries, the specified antigen can be used to immunize a non-human animal, for example, a rodent, for example a mouse, hamster or rat. In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, it is possible to genetically modify mouse strains lacking production of mouse antibodies with large fragments of human Ig loci. Using the hybridoma technology, antigen-specific mono-clonal antibodies derived from genes with the desired specificity can be produced and selected. See, for example, XENOMOUSE ™, Green et al. (1994) Nature Genetics 7: 13-21, US 2003-0070185, WO 96/34096, published October 31, 1996, and PCT Application No. PCT / US96 / 05928, filed on April 29, 1996.

In another embodiment, a UADS molecule from a non-human animal is obtained and then modified, eg, humanized, deimmunized, chimeric, can be produced using recombinant DNA techniques known in the art. Various strategies have been described for making chimeric antibodies and UADS molecules. See, for example, Morrison et al., Proc. Nati Acad. Sci. U.S. A. 81: 6851, 1985; Takeda et al., Nature 314: 452, 1985, Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Pat. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B. also can humanized antibodies and UADS molecules are produced, for example, using transgenic mice expressing heavy and light human chain genes, but which can not express the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR grafting procedure that can be used to prepare the humanized antibodies and UADS molecules described herein (U.S. Patent No. 5,225,539). All CDRs of a particular human antibody can be substituted with at least one part of a non-human CDR or only some CDRs can be substituted with non-human CDRs. It is only necessary to replace the number of CDRs necessary for the binding of humanized antibody and UADS molecule to a predetermined antigen.

Humanized antibodies can be generated by substituting variable domain Fv sequences that are not directly involved in antigen binding with equivalent sequences of human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229: 1202-1207; by Oi et al. (1986) BioTechniques 4: 214; and by US 5,585,089 documents; US 5,693,761; US 5,693,762; US 5,859,205; and US 6,407,213. These methods include isolating, manipulating and expressing the nucleic acid sequences encoding all or part of the immunoglobulin Fv variable domains from at least one heavy or light chain. Said nucleic acids can be obtained from a hybridoma by producing a nanobody against a predetermined target, as has been described above, as well as from other sources. The recombinant DNA encoding the humanized UADS molecule, for example nanobody molecule, can then be cloned into an appropriate expression vector.

In some embodiments, a humanized UADS molecule, e.g., nanobody molecule, is optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and / or retromutations. Said modified immunoglobulin molecules can be manufactured by any of various techniques known in the technical field (for example, Teng et al., Proc. Nati, Acad. Sci. USA, 80: 7308-7312, 1983; Kozbor et al., Immunology. Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982) and can be manufactured in accordance with the teachings of PCT publication W092 / 06193 or EP 0239400).

Techniques for humanizing UADS molecules, for example nanobody molecules, are disclosed in WO 06/122786.

A UADS molecule, eg, nanobody molecule, can also be modified by specific deletion of human T lymphocyte epitopes or "deimmunization" by the methods described in WO 98/52976 and WO 00/34317. In summary, the heavy and light chain variable domains of, for example, a nanobody can be analyzed to determine peptides that bind to Class II MHC; these peptides represent possible T lymphocyte epitopes as defined in WO 98/52976 and WO 00/34317). For the detection of possible epitopes of T lymphocytes, a computerized modeling strategy called "peptide striation" can be applied and, furthermore, motifs present in the VH and VL sequences can be searched in a database of peptides binding to the human MHC class II. as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major DR alotypes of MHC class II and thus constitute possible epitopes of T lymphocytes. The possible detected T lymphocyte epitopes can be eliminated by replacing few amino acid residues in the variable domains or, preferably, by simple amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, a common amino acid can be used at a position in the human germline antibody sequences. The sequences of the human germ line, for example, are disclosed in Tomlinson, et al. (1992) J. Mol. Biol. 227: 776-798; Cook, G. P. et al. (1995) Immunol. Today Vo. 16 (5): 237-242; Chothia, D. et al. (1992) J. Mol. Biol. 227: 799-817; and Tomlinson et al. (1995) EMBOJ. 14: 4628-4638. The V BASE directory provides a comprehensive directory of variable regions of human immunoglobulin sequences (compiled by Tomlinson, I.A., et al., MRC Center for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for conserved framework regions and CDRs. Consensus human framework regions can also be used, for example, as described in US 6,300,064.

UADS molecules, for example, nanobody molecules, can be produced by living host cells that have been genetically modified to produce the protein. Methods for genetically modifying cells to produce proteins are well known in the art. See, for example, Ausabel et al., Eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Said methods include the introduction of nucleic acids that encode and allow the expression of the protein in living host cells. These host cells can be bacterial cells, fungal cells or preferably, cells of animals that grow in culture. Bacterial host cells include, but are not limited to, Escherichia coli cells. Examples of suitable E. coli strains include: HB101, DH5a, GM2929, JM109, KW251. NM538, NM539, and any E. coli strain that does not excise foreign DNA. Fungal host cells that can be used include, but are not limited to, cells of Saccharomyces cerevisiae, Pichia pastoris and Aspergillus. Few examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3 and WI38. Using procedures well known to those skilled in the art, new animal cell lines can be established (eg, by transformation, viral infection and / or selection). Optionally, the host cells can secrete the protein in the medium.

Modified UADS molecules The formulations of the present invention may contain at least one UADS molecule, for example, nanobody molecule, having an amino acid sequence that differs at least at an amino acid position in one of the framework regions conserved from the amino acid sequence of domain of natural origin, for example, VH domain.

It should be understood that the amino acid sequences of some of the UADS molecules of the present invention, such as humanized UADS molecules, may differ in at least one amino acid position in at least one of the framework regions conserved from the amino acid sequences of domain of natural origin, for example, VHI-I domains of natural origin.

The present invention also includes formulations of derivatives of the UADS molecules. Said derivatives can generally be obtained by modification and in particular by chemical and / or biological (for example enzymatic) modification, of the UADS molecules and / or of one or more of the amino acid residues that form the UADS molecules described herein. .

Examples of such modifications, as well as examples of amino acid residues within the sequence of the UADS molecule that can be modified in such a way (ie on the framework of the protein but preferably in a side chain), procedures and techniques that can be used to introduce such modifications and possible uses and Advantages of said modifications will become obvious to the expert in the field.

For example, such modification may involve the introduction (for example by covalent bond or other suitable manner) of one or more functional groups, residues or residues in or on the UADS molecule and in particular one or more functional groups, residues or residues. that confer one or more desired properties or functionalities to the UADS molecules. Examples of such functional groups will be obvious to one skilled in the art.

For example, said modification may comprise the introduction (eg, by covalent attachment or in any other suitable manner) of one or more functional groups that increase the half-life, solubility and / or absorption of the UADS molecule, which reduces the immunogenicity and / or toxicity of the UADS molecule, which eliminates or attenuates any of the undesirable side effects of the UADS molecule and / or which confers other advantageous properties to and / or reduces the undesired properties of the UADS molecule; or any combination of two or more of the above. Examples of such functional groups and / or methods for introducing them will be apparent to the person skilled in the art and can generally comprise all functional groups and methods mentioned in the general prior art and cited hereinabove as well as functional groups and procedures. known in themselves for the modification of proteins pharmaceuticals and in particular for the modification of antibodies or antibody fragments (including ScFvs and 148 single domain antibodies), the reference of which is made, for example, in Remington's Pharmaceutical Sciences, 16th edition., Mack Publishing Co., Easton, PA (1980). Said functional groups can be linked, for example, directly (for example covalently) to a Nanobody of the present invention or optionally by means of a suitable linker or spacer, as will again be apparent to the person skilled in the art.

One of the widely used techniques for increasing the half-life and / or reducing the immunogenicity of the pharmaceutical proteins comprises the adhesion of a suitable pharmacologically acceptable polymer, such as poly (ethylene glycol) (PEG) or derivatives thereof (such as methoxypoly (ethylene glycol)). or mPEG). Generally, any suitable form of pegylation, such as pegylation used in the art, can be used for antibodies and antibody fragments (including but not limited to domain antibodies (single) and ScFvs); reference is made, for example, in Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and WO 04/060965. Various reagents for protein pegylation are also available on the market, for example from Nektar Therapeutics, United States.

Preferably, directed pegylation is used, in particular by a cysteine residue (see, for example, Yang et al., Protein).

Engineering, 16, 10, 761-770 (2003). For example, for this purpose, the PEG can bind to a cysteine residue that occurs naturally in a UADS molecule, a UADS molecule can be modified so that one or more cysteine residues are properly introduced to bind to PEG or an amino acid sequence comprising one or more cysteine residues for binding to PEG can be fused to the N and / or C terminus of a Nanobody of the present invention, all using methods of genetic modification of proteins known per se by the skilled artisan. in the matter.

Preferably, for the UADS molecule, a PEG with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000.

With respect to pegylation, it should be noted that generally, the present invention also includes any UADS molecule that has been pegylated at one or more amino acid positions, preferably such that said pegylation (1) increases the half-life in vivo; (2) reduce immunogenicity; (3) provides one or more additional beneficial properties known per se for pegylation; (4) does not substantially influence the affinity of the UADS molecule (for example it does not reduce said affinity by more than 90%, preferably not by more than 50% and not by more than 10%, as determined by a suitable assay, as described in the Examples below); and / or (4) does not influence none of the other desired properties of the UADS molecule. The PEG groups and methods for joining them, both specifically and non-specifically, will be apparent to the person skilled in the art.

In addition, normally less preferred modifications comprise N-linked or O-linked glycosylation, typically as part of co-translational and / or post-translational modification, depending on the host cell used for the expression of the UADS molecule.

Formulations A formulation of a UADS molecule, eg, nanobody molecule, includes a UADS molecule, a compound that can serve as a cryoprotectant and a buffer. The pH of the formulation is generally pH 5.5 - 7.0. In some embodiments, a formulation is preserved as a liquid. In other embodiments, a formulation is prepared as a liquid and then dried, for example, by lyophilization or spray drying, before storage. A dry formulation can be used as a dry compound, for example, as an aerosol or powder or reconstituted to its original or other concentration, for example, using water, a buffer, or other suitable liquid.

The purification process of the UADS molecule is designed to allow the transfer of the UADS molecule into a suitable formulation for long-term storage as a frozen liquid and subsequently for freeze drying (for example, using a histidine / sucrose formulation). The formulation is lyophilized with the protein at the specific concentration. The lyophilized formulation can then be reconstituted as necessary with a suitable diluent (e.g., water) to re-solubilize the original formulation components at a desired concentration, generally the same or higher concentration as compared to the concentration prior to lyophilization.

The lyophilized formulation can be reconstituted to produce a formulation having a concentration that differs from the original concentration (i.e., prior to lyophilization), depending on the amount of water or diluent added to the lyophilizate with respect to the volume of liquid that was originally dried by freezing Suitable formulations can be identified by testing one or more parameters of the integrity of the antibodies. The parameters tested are generally the percentage of APM species or the percentage of BPM species.

The percentage of APM species or BPM species is determined as a percentage of the total protein content in a formulation or as a change in percentage increase over time (ie, during storage). The total percentage of APM species in an acceptable formulation is not more than 10% of APM species after storage as a lyophilisate or liquid at -20 ° C to 40 ° C (for example, from -20 ° C to 25 ° C, from -20 ° C to 15 ° C, from 2 ° C to 8 ° C, to approximately 2 ° C, or to approximately 25 ° C) for at least one year or not more than approximately 10% of BPM species after storage as a lyophilized or liquid of -20 ° C to 40 ° C for at least one year. "Approximately" refers to + 20% of a numerical value quoted. Therefore, "approximately 20 ° C" means from 16 ° C to 24 ° C.

Typically, the stability profile is less than 10% APM / BPM of 2 ° - 8 ° C for a refrigerated product and 25 ° C for a product at room temperature. The APM species or the BPM species are tested in a formulation preserved as a lyophilisate after reconstitution of the lyophilizate. The temperature of 40 ° C is an accelerated state which is generally used to test stability and determine stability during short-term exposures with respect to non-preservation conditions, for example, as may occur during the transfer of a product during transport .

When the parameter tested is the percentage change of APM species or BPM species, the percentage of total protein in one or both species after storage is compared to the percentage of total protein in one or both species before storage (eg, after of the preparation of the formulation). The difference in percentages is determined. In general, the protein percentage change of APM species or BPM species in liquid formations is not more than 10%, for example, not greater than about 8%, not more than about 7%, not more than about 6%, no more than about 5%, no more than about 4%, or no more than about 3% after storage at 2 ° C - 82 ° C or 25 ° C for approximately 18 to 24 months. "Approximately" means ± 20% of a numerical value quoted, typically, within 10% and more typically, within 5% of a given value or range of values. Therefore, approximately 10% means from 8% to 12%. Formulations stored as lyophilized product generally have less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of species of APM or less than about 5%, less than about 4%, less than about 3% or less than about 2% or less than about 1% of BPM species after reconstitution or in a liquid formulation, after storage at - 30 ° C - 8 ° C (for example, 4 ° C or -20 ° C) for approximately six, nine, ten, twelve, fifteen, eighteen to twenty-four months.

Formulations of UADS molecules (e.g., TNF-binding nanobody molecules) can be stored as a frozen liquid formulation or as a lyophilized, for, for example, at least six, nine, ten, twelve months or at least two years , at least three years, at least four years or at least five years. In one example, a formulation of the TNF-binding nanobody molecule contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 50 mg / ml TNF-binding nanobody molecules and has a pH of 6.0. In another example, the TNF-binding nanobody molecule formulation contains 20 mM histidine, saccharose ai 7.5%, polysorbate 80 0.01%, nanobody molecules binding to TNF 50 mg / ml and has a pH of 6.0. In another example, the formulation contains 20 mM histidine, 10% sucrose, 0.02% Polysorbate 80, 100 mg / ml TNF binding nanobody molecule and has a pH of 6.0. In another example, the formulation contains 10 mM histidine, 5% sucrose, 50 mg / ml TNF-binding nanobody molecule and has a pH of 6.0. In yet another example, the formulation contains 20 mM histidine, 0% sucrose, TNF binding nanobody 100 mg / ml and has a pH of 6.0. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, approximately 80 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In yet another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 100 mM Arginine (base), 88 to 100 mg / ml TNF binding nanobody molecule and has a pH of 5.8. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 55 mM NaCl, 88 to 100 mg / ml of TNF-binding nanobody molecule and has a pH of 6.1. In yet another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 55 mM Arginine HCI, 88 to 100 mg / ml of TNF-binding nanobody molecule and has a pH of 6.1. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 100 mM Glycine, 88 to 100 mg / ml of TNF binding nanobody molecule and has a pH of 6.0. In another additional example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 100 mM methionine, from 88 to 100 mg / ml of TNF-binding nanobody molecule, and has a pH of 6.0. In another example, the formulation contains 10 mM histidine, 8% sucrose, 0.01% Polysorbate 80, 88 to 100 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In yet another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 88 to 100 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In another example, the formulation contains 20 mM Histidine, 5% Sucrose, 118 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In yet another example, the formulation contains 20 mM Tris, 5% Sucrose, 117 mg / ml of TNF-binding nanobody molecule and has a pH of 7.2. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, approximately 80 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In yet another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, approximately 50 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In one example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, about 1 mg / ml of TNF-binding nanobody molecule and has a pH of 5.5. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginine HCI, about 1 mg / ml of TNF-binding nanobody molecule and has a pH of 5, 5. In yet another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mM sodium chloride, approximately 1 mg / ml of TNF-binding nanobody molecule and has a pH of 5.5. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, about 1 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In another example, the formulation contains 0 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginine HCI, about 1 mg / ml of TNF-binding nanobody and has a pH of 6.0. In yet another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mM sodium chloride, about 1 mg / ml of TNF-binding nanobody molecule and has a pH of 6.0. In one example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, about 1 mg / ml of TNF-binding nanobody molecule and has a pH of 6.5. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginine HCI, about 1 mg / ml of TNF-binding nanobody molecule and has a pH of 6.5. In yet another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mM sodium chloride, approximately 1 mg / ml of TNF-binding nanobody molecule and has a pH of 6.5 . In one example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, about 1 mg / ml of TNF-binding nanobody molecule, and has a pH of 7.0. In another example, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginine HCI, about 1 mg / ml of TNF-binding nanobody molecule and has a pH of 7.0. In another example In addition, the formulation contains 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mM sodium chloride, approximately 1 mg / ml of TNF-binding nanobody molecule and has a pH of 7.0. In yet another example, the formulation of the TNF-binding nanobody molecule contains 20 mM histidine, 7.5% sucrose, 0.01% Polysorbate 80, 250 mg / ml of TNF-binding nanobody molecules and has a pH of 6.0 .

Subsequently, additional details related to components of formulations and methods for testing the integrity of the UADS molecule are provided, for example, the TNF-binding nanobody molecule in a formulation.

In the formulations, the concentrations of the UADS molecule are generally between about 0.1 mg / ml and about 350 mg / ml, eg, 0.5 mg / ml to about 350 mg / ml, of about 0.5 mg / ml at about 300 mg / ml, from about 0.5 mg / ml to about 250 mg / ml, from about 0.5 mg / ml to about 150 mg / ml, from -about 1 mg / ml to about 130 mg / ml, about 10 mg / ml to about 130 mg / ml, from about 50 mg / ml to about 120 mg / ml, from about 80 mg / ml to about 120 mg / ml, from about 88 mg / ml to about 100 mg / ml , or about 10 mg / ml, about 25 mg / ml, about 50 mg / ml, about 80 mg / ml, about 100 mg / ml, about 130 mg / ml, about 150 mg / ml, about 200 mg / ml, about 250 mg / ml or about 300 mg / ml. In the context of intervals, "approximately" means - 20% of the value of the numerical range cited lower and + 20% of the value of the numerical interval mentioned higher. In the context of ranges, for example, from about 10 mg / ml to about 100 mg / ml, it refers to between 8 mg / ml and 120 mg / ml. In some cases, the concentrations of the UADS molecule in the formulations can be, for example, between 0.1 mg / ml and 200 mg / ml, for example, 0.5 mg / ml and 100 mg / ml, 0.5 mg / ml and 1 , 0 mg / ml, 0.5 mg / ml and 45 mg / ml, 1 mg / ml and 10 mg / ml, 10 mg / ml and 40 mg / ml, 10 mg / ml and 50 mg / ml, 50 mg / ml and 100 mg / ml, 100 mg / ml and 200 mg / ml. Said formulations of UADS molecules can be used as therapeutic agents. Therefore, the concentration of the UADS molecule in a formulation is sufficient to provide said dosages in a volume of the formulation that tolerates the subject to be treated and is appropriate for the method of administration. In a non-limiting example, to provide a high dosage subcutaneously, in which the volume limitation is small, (for example, from about 1 ml to 1.2 ml per injection), the concentration of the UADS molecule is generally at least 100 mg / ml or higher, for example, from 100 mg / ml to 500 mg / ml, from 100 mg / ml to 250 mg / ml or from 100 mg / ml to 150 mg / ml. Such high concentrations can be achieved, for example, by reconstituting a lyophilized formulation in a suitable volume of diluent (eg, sterile water for injection, buffered saline). In some cases, the reconstituted formulation has a concentration of between about 100 mg / ml and 300 mg / ml (eg, 100 mg / ml, 125 mg / ml, 150 mg / ml, 175 mg / ml, 200 mg / ml , 250 mg / ml, 275 mg / ml, 300 mg / ml). For long-term storage, high concentrations, for example up to 250 mg / ml, for example, frozen storage of large preparations of the UADS molecule can be used.

For administration by inhalation, the formulation is generally somewhat concentrated (eg, between about 100 mg / ml and 500 mg / ml) to provide a sufficient dose in a limited volume of aerosol for inspiration. In some cases, low concentrations are used (e.g., between about 0.05 mg / ml and 1 mg / ml). Methods for adapting the dosage administered with respect to the method of administration, for example, a jet nebulizer or a metered dose aerosol, are known in the art.

Buffers and cryoprotectants The pH of a formulation as described herein is generally from about pH 5.0 to about 7.0, for example, pH from about 5.5 to about 6.5, pH from about 5.5 to about 6.0, pH from about 6.0 to about 6.5, pH 5.5, pH 6.0 or pH 6.5. In general, a buffer that can maintain a pH solution of 5.5 to 6.5 to prepare a formulation, for example, a buffer having a pKA of about 6.0. Suitable buffers include, without limitation, histidine, TRIS, 2- (N-morpholino) ethanesulfonic acid (MES) buffer, cacodylate, phosphate, acetate, succinate and citrate. The concentration of the buffer is between about 4 mM and about 60 mM, for example, from about 5 mM to about 25 mM, for example, histidine is generally used at a concentration of up to 60 mM. In some cases, the histidine buffer is used at a concentration of approximately 5 mM, approximately 10 mM or approximately 20 mM. In some cases, acetate or succinate buffer at a concentration of about 5 mM or about 10 mM is used.

Cryoprotectants are known in the art and include, for example, sucrose, trehalose and glycerol. Generally, a cryoprotectant that shows low toxicity in biological systems is used. The cryoprotectant is included in the formulation at a concentration of from about 0.5% to 15%, from about 0.5% to 2%, from about 2% to 5%, from about 5% to 10%, of approximately 10% to 15% and approximately 5% (weight / volume).

Histidine buffer, which can be used as a buffer in a TNF-binding nanobody formulation, can have cryoprotective properties. In some embodiments of the invention, a histidine buffer is used together with a cryoprotectant such as a sugar, for example, saccharose. A formulation of the present invention can specifically exclude the use of histidine in any substantial amount, for example, neither the buffer nor the cryoprotective component of the formulation is a histidine.

The viscosity of a formulation is generally one that is compatible with the route of administration of the formulation. In some embodiments, the viscosity of the formulation is between 1 cP and 4 cP, for example, from about 2 cP to 3.5 cP. In other embodiments, the viscosity of the formulation is between about 5 cP and about 40 cP. In specific embodiments, the viscosity of the formulation is about 1 cP, 2 cP, 2.4 cP at 2.8 cP, 3 cP, 3.1 cP at 3.2 cP, 4 cP, 5 cP, 10 cP, cP, 20 cP, 25 cP, 30 cP, 35 cP or 40 cP.

Surfactants In some embodiments, a surfactant is included in the formulation. Examples of surfactants include, without limitation, nonionic surfactants such as polysorbates (e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80 or polysorbate-85); Triton ™; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulphobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl- Betaine, Linoleamidopropyl betaine, Myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine (for example lauroamidopropyl), myristinidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; methyl cocoyl sodium or disodium methyl ofeil-taurate and the Monaquat ™ series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol, polypropylene glycol and copolymers of ethylene and propylene glycol for example, poloxamers (for example, poloxamer 188).

The amount of surfactant added is such that it reduces the aggregation of the reconstituted protein to an acceptable level as tested, using, for example, HPLC-SEC of APM species or DPM species and minimizes particle formation after reconstitution of a lyophilized of a TNF-binding nanobody formulation. It has also been shown that surfactant addiction reduces the reconstitution time of a lyophilized formulation of TNF-binding antibodies and aids in degassing the solution. For example, the surfactant may be present in the formulation (liquid or prior to lyophilization) in an amount of about 0.001% to 0.6%, eg, from about 0.005% to 0.05%, from about 0.005% to about 0.2% and from approximately 0.01% to 0.2%.

Additions to the Formulations The formulations are stored as sterile or lyophilized sterile solutions. The prevention of the action of microorganisms in the formulations can also be achieved by including at least one agent antibacterial and / or antifungal in a formulation, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In some cases, a lyophilizate is reconstituted with bacteriostatic water (for example, water containing 0.9% benzyl alcohol). Techniques for the inclusion of a preservative in a formulation as well as methods for identifying preservatives that are compatible with a specific formulation and administration process are known in the art (for example, see Gupta, et al. (2003), AAPS Pharm. 5: article 8, pages 1-9).

In some cases, the formulation is isotonic. In general, any component known in the art that contributes to the osmolarity / tonicity of the solution can be added to a formulation (for example, salts, sugars, polyalcohols or a combination thereof). Isotonicity is generally achieved by using a component of a basic formulation (such as sucrose) in an isotonic concentration or by adding an additional component, such as a sugar, a polyalcohol such as mannitol or sorbitol or a salt such as sodium chloride.

In some cases a salt is used in a TNF-binding nanobody formulation, for example, to achieve isotonicity or to increase the integrity of the TNF-binding nanobody of the formulation. Salts suitable for use are described, above. The salt concentration can be from about 0 mM to about 300 mM.

In some cases, the formulation is prepared with Tween (for example Tween® 20, Tween® 80) to decrease interfacial degradation. The concentration of Tween can be from about 0.001% to about 0.05%. In one example, Tween 80 is used at a concentration of 0.01% in the formulation.

In other determined cases, the formulation is prepared with arginine. The concentration of arginine in the formulation can be from about 0.01% to about 5%. In one example, arginine is used at a concentration of 2% in the formulation. In some cases Tween and arginine are added to the TNF binding formulations described herein.

In still other cases, the formulation can be prepared at least one of: sorbitol, glycine, methionine or sodium chloride. If sorbitol is included in the formulation, it may be added at a concentration of between about 1% and about 10%. In one example, sorbitol is found in the formulation at a concentration of 5%. If glycine is included in the formulation, this can be added at a concentration of between about 0.1% to about 2%. In one example, glycine is observed in the formulation at a concentration of 1%. If methionine is included in the formulation, it may be added at a concentration of between about 5 mM and about 150 mM. In one example, methionine is added in the formulation at a concentration of 00 mM. In another example, methionine is added to the formulation at a concentration of about 10 mM, about 20 mM or about 70 mM. If sodium chloride is included in the formulation, it may be added at a concentration of between about 5 mM and about 100 mM. In one example, sodium chloride is added to the formulation at a concentration of 55 mM.

Storage and Preparation Procedures Freezing In some cases, formulations containing antibody are frozen for storage. Accordingly, it is desirable that the formulation be relatively stable under such conditions, including freeze / thaw cycles. One method to determine the suitability of a formulation is to subject a sample formulation to at least two, for example, three, four, five, eight, ten or more freezing cycles (at, for example, -20 ° C or -80 ° C) and thawing (for example by rapid thawing in a hot bath at 37 ° C or slow thawing at 2o - 8 ° C), determining the amount of APM species and / or BPM species that accumulate after freezing cycles / thawing and comparing them with the amount of APM species or BPM species present in the sample before the freeze-thaw procedure. An increase in APM or BPM species indicates that stability decreases.

Lyophilization The formulations can be stored after lyophilization. Therefore, it is useful to test a formulation to determine the stability of the protein component of the formulation after lyophilization to determine the suitability of a formulation. The procedure is similar to that described above for freezing, except that the sample formulation is lyophilized instead of frozen, reconstituted to its original volume and tested for the presence of APM species and / or BPM species. The formulation of the lyophilized sample is compared to a corresponding sample formulation that has not been lyophilized. An increase in the APM or BPM species in the lyophilized sample compared to the corresponding sample indicates decreased stability in the lyophilized sample.

In general, a lyophilization protocol includes loading a sample in a lyophilizer, a period of pre-cooling, freezing, vacuum initiation, leveling up to the first drying temperature, primary drying, leveling up to the second drying temperature, second drying, and stop the sample. Additional parameters that can be selected for a lyophilization protocol include vacuum (e.g., in microns) and condensation temperature. Suitable leveling rates for temperature are between approximately 0.1 ° C / min at 2 ° C / min, for example, 0.1 ° C / min at 1.0 ° C / min, from 0.1 ° C / min at 0.5 ° C / min, 0.2 ° C / min at 0.5 ° C / min, 0.1 ° C / min, 0.2 ° C / min, 0.3 ° C / min, 0.4 ° C / min, 0.5 ° C / min, 0.6 0C / min, 0.7 ° C / min, 0.8 ° C / min, 0.9 ° C / min and 1.0 ° C / min. Valid temperatures during freezing for a lyophilization cycle are generally from about -55 ° C to -5 ° C, from -25 ° C to -5 ° C, from -20 ° C to -5 ° C, from - 15 ° C to -5 ° C, -10 ° C to -5 ° C, -10 ° C, -1 1 ° C, -12 ° C, -13 ° C, -14 ° C, -15 ° C , -16 ° C, -17 ° C, -18 ° C, -19 ° C, -20 ° C, -21 ° C, -22 ° C, -23 ° C, -24 ° C or -25 ° C . Valid temperatures may be different from the first and second drying, for example, the first drying may be performed at a temperature lower than the second drying. In a non-limiting example, the first drying can be carried out at 0 ° C and a secondary drying at 25 ° C.

In some cases, a reheat protocol is used during freezing and before starting the vacuum. In such cases, the reheat time must be selected and the temperature is generally above the glass transition temperature of the composition. In general, the reheat time is about 2 to 15 hours, about 3 to 12 hours, about 2 to 10 hours, about 3 to 5 hours, about 3 to 4 hours, about 2 hours, about 3 hours, approximately 5 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours or approximately 15 hours. The temperature for reheating is generally from about -35 ° C to about -5 ° C, for example from about -25 ° C to about -8 ° C, from about -20 ° C to about -10 ° C.

° C, of about -25 ° C, of about -20 ° C, of about -15 ° C, of about 0 ° C or about -5 ° C. In some cases, the reheat temperature is generally -35 ° C to 0 ° C, for example, -25 ° C to -8 ° C, -20 ° C to -10 ° C, -25 ° C, -20 ° C, -15 ° C, 0 ° C.

The stability of the formulations described herein can be tested using a variety of lyophilization parameters including: the first valid drying temperatures of -25 ° C to 30 ° C and secondary drying durations of 2 hours to 9 hours at 0 ° C at 30 ° C.

In a non-limiting example, a formulation of 10 mM histidine, 5% sucrose, 0.01% polysorbate 80, pH 6.0 at a protein concentration of 50 mg / ml of TNF-binding nanobody was formulated in bulk and lyophilized. After lyophilization, the product was reconstituted with about half the loading volume to deliver the protein at 100 mg / ml. It was shown that the TNF antibody was strong after lyophilization to extremes at product temperatures. The stability profile after storage at 50 ° C for 4 weeks was identical for material that had been prepared using a variety of lyophilization cycles (for example, see Figures 16-20), some of which had differences of almost 10 ° C in the product temperature during primary drying (for example, Figure 13). In general, a lyophilization cycle can be carried out from 10 hours to 100 hours, for example, 20 hours to 80 hours. hours, 30 hours to 60 hours, 40 hours to 60 hours, 45 hours to 50 hours, 50 hours to 65 hours.

The delimiting examples of the temperature range for the storage of an antibody formulation is from about -20 ° C to about 50 ° C, for example, from about -15 ° C to about 30 ° C, from about -15 ° C to about 20 ° C, from about 5 ° C to about 25 ° C, from about 5 ° C to about 20 ° C, from about 5 ° C to about 15 ° C, from about 2 ° C to about 12 ° C, about 2 ° C to about 10 ° C, about 2 ° C to about 8 ° C, about 2 ° C to about 6 ° C or about 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 10 ° C, 15 ° C, 25 ° C or 30 ° C. In some cases, regardless of the storage temperatures, the samples are stable to temperature changes that may occur transiently during the storage and transport conditions of what could be foreseen for said compositions.

Drying by Spraying In some cases, a formulation is spray dried and then stored. Spray drying is performed using methods known in the art and can be modified to use liquid or frozen spray drying (e.g., using procedures such as those of Niro Inc. (Madison, WI), Upperton Particle Technologies (Nottingham, England), or Buchi (Brinkman Instruments Inc., Westbury, NY), or US Patent Publication Nos. 20030072718 and 20030082276).

Determination of the Integrity of the UADS Molecule The accumulation of APM species and BPM species are useful measures of antibody instability. The accumulation of APM or BPM in a formulation is indicative of the instability of a protein stored as part of the formulation. HPLC chromatography by size exclusion can be used to determine the presence of APM and BPM species. Suitable systems for such measurements are known in the art, for example HPLC systems (Waters, Milford, MA). Other known systems for evaluating the integrity of the antibodies in a formulation can be used in the art, for example, SDS-PAGE (to control APM and BPM species), bioassays of antibody activity, enzyme-linked immunosorbent assay, ability to bind proteins purified targets (for example, TNFa) and cation exchange HPLC (HPLC-CEX, to detect variants and control surface changes). In one example, a bioassay is a cell-based assay in which the inhibition of TNFα-dependent activity is examined in the presence of different concentrations of the nanobody molecule formulated to demonstrate biological activity.

Manufacturing Articles The present application also provides an article of manufacture that includes a formulation as described herein and provides instructions for use in the formulation.

The formulations to be used to administer to a subject, for example, as a pharmaceutical agent must be sterile. This can be achieved using methods known in the art, for example, by filtration through sterile filtration membranes, before, or after, the formulation of a liquid or lyophilization and reconstitution. Alternatively, when this does not damage the structure, the components of the formulation can be sterilized using autoclave and then combined with components sterilized by radiation or filter to produce the formulation.

In the pharmaceutical formulation it can be administered with a transcutaneous delivery device, such as a syringe, which includes a hypodermic or multi-chamber syringe. In one embodiment, the device is a pre-filled syringe in which a needle is incorporated or integrated. In other embodiments, the device is a pre-filled syringe that does not have a needle incorporated. The needle can be packaged with the previously loaded syringe. In one embodiment, the device is an autoinjector device, for example, a self-injecting syringe. In another embodiment, the injection device is an injection pen. In still another embodiment, the syringe is a syringe with needle on stake, luer lock syringe or luer slip syringe. Other suitable delivery devices include stents vascular, catheter, microneedle and implantable controlled release devices. The composition can be administered intravenously with conventional IV equipment, including, for example, IV tubing catheters, with or without in-line filters.

In some embodiments, a syringe is suitable for use with an autoinjector device. For example, the autoinjector device may include a simple vial system, such as pen injector device for supplying a solution. Such devices are available on the market from manufacturers such as BD Pens, BD Autojector®, Humaject®, NovoPen®, BD®Pen, AutoPen® and OptiPen®, GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen ®, Roferon Pen®, Biojector®, Iject®, J-tip Needle-Free Injector®, DosePro®, Medi-Ject®, for example, as manufactured or developed by Becton Dickensen (Franklin Lakes, NJ), Ypsomed (Burgdorf) , Switzerland, www.ypsomed.com, Bioject, Portland, Oregon, National Medical Products, Weston Medical (Peterborough, United Kingdom), Medi-Ject Corp (Minneapolis, Minn.), And Zogenix, Inc, Emeryville, CA. Recognized devices comprising a dual road system include the pen injectors systems for reconstituting a lyophilized drug in a cartridge for delivery of the reconstituted solution such as HumatroPen®.

The article of manufacture may include a container suitable for containing the formulation. A suitable container can be, without limitation, a device, vial, vial, syringe, test tube, nebulizer (e.g. ultrasonic or vibration mesh nebulizers), solution bag i.v. or inhaler (for example, a metered dose inhaler (MDI) or dry powder inhaler (IPS)). The package can be formed by any suitable material such as glass, metal or a plastic such as polycarbonate polystyrene or polypropylene. For example, the package (eg, syringe or vial) can be made with glass, plastic or a cyclic olefin copolymer or a cyclic olefin polymer. Optionally, the package (eg, syringe or vial) has a stopper, for example, a rubber stopper. Specific embodiments of packages for storing the present formulations include: (i) liquid in a glass vial with a rubber stopper; (ii) liquid in a syringe previously filled with glass with a rubber plunger; and (iii) liquid in a pre-charged polymer syringe, for example cyclic olefin copolymer (COC) or cyclic olefin polymer (COP), with rubber plunger.

In general, the package is made of a material that does not absorb significant amounts of protein from the formulation and does not react with the components of the formulation.

In some embodiments, the package is a clear glass vial with a stopper, for example, a gray silicone stopper West 4432/50 1319 or a Durafluor West 4023 stopper. In some embodiments, the package is a syringe. In specific embodiments, the formulation comprises 100 mg / ml of the TNF-binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01% polysorbate-80, pH 6.0 in a pre-filled syringe. In another embodiment, the formulation comprises approximately 10 mg / ml, approximately 100 mg / ml of the TNF binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01% polysorbate-80, pH 6 in a prefilled cyclic olefin syringe and a gray rubber plunger of silicone West 4432/50. In other embodiments, the formulations include about 10 mg / ml, about 50 mg / ml, about 100 mg / ml of the TNF-binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01% polysorbate-80 , pH 6 in a pre-loaded glass syringe and a West 4432/50 gray silicone plunger or West 4023/50 rubber plunger coated with Flourotec / B2 Daikyo.

The articles of manufacture described herein may additionally include a packaging material. The packaging material provides, in addition to information for use or administration, for example, information needed by a regulatory agency regarding the conditions under which the product may be used. For example, the packaging material can provide instructions to the patient how to inject a pre-filled syringe containing the formulations described herein or how to reconstitute the lyophilized formulation in an aqueous diluent to form a solution in a specified period, for example, for a period of 2-24 hours or more. The currently claimed formulations are useful for the use of the pharmaceutical in humans.

In some embodiments, the formulations may be administered as nebulizers. Examples of nebulizers include, in non-limiting examples, jet nebulizers, ultrasonic nebulizers and mesh nebulizers with vibration. These classes use different procedures to create an aerosol from a liquid. In general, any device that generates an aerosol that can preserve the integrity of the protein in these formulations is suitable for the administration of formulations as described herein.

In other embodiments, the pharmaceutical compositions can be administered with medical devices. For example, the pharmaceutical compositions can be administered with a needleless hypodermic injection device, such as the devices described in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941, 880, 4,790,824 or 4,596,556. Examples of well-known implants and modules include: U.S. Patent No. 4,487,603 which discloses an implantable microinfusion pump for dispensing medication at a controlled rate: U.S. Patent No. 4,486,194, which describes a therapeutic device for administer medications through the skin; U.S. Patent No. 4,447,233, which discloses a medication pump for administering medication at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses an implantable variable flow infusion apparatus for continuous administration of the drug; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments and U.S. Patent No. 4,475,196 which describes a system of administration of osmotic drug. The therapeutic composition may also be in the form of a biodegradable or non-biodegradable sustained release formulation for subcutaneous or intramuscular administration. See, for example, U.S. Patent Nos. 3,773,919 and 4,767,628 and PCT Application No. WO 94/15587. Continuous administration can also be achieved using an implantable or external pump. The administration can also be carried out intermittently, for example, single daily injection or continuously at a low dose, for example, prolonged release formulation. The delivery system can be modified to optimally adjust to the administration of the UADS molecule. For example, a silicone syringe can be manufactured to the extent that it is optimal for storage and delivery of the UADS molecule. Of course, many other such implants, delivery systems and modules are also known. The invention also features a device for administering a first and second agent. The device can include, for example, one or more casings for storing pharmaceutical preparations and can be configured to deliver unit doses of the first and second agents. The first and second agents can be stored in the same compartment or individual. For example, the device can combine the agents before administration. It is also possible to use different devices to administer the first and second agents.

Administration and Treatment Procedure The formulations of the present invention can be administered to a subject (e.g., a human subject) alone or in combination with a second agent, e.g., a second therapeutic or pharmacologically active agent, to treat or prevent (e.g., reducing or improving one or more of the symptoms associated with) a disorder associated with TNFa, for example, inflammatory or autoimmune disorders. The term "treatment" refers to administering a therapy in an amount, manner and / or manner effective to ameliorate a condition, symptom or parameter associated with a disorder or to prevent the progression of a disorder to a statistically significant degree or to a detectable degree. for an expert in the field. An effective amount, manner or mode may vary depending on the subject and may be adjusted to the subject.

Non-limiting examples of immunological disorders that can be treated include, but are not limited to, autoimmune disorders, for example, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, arthritis associated with lupus or ankylosing spondylitis), scleroderma, systemic lupus erythematosus, Sjogren's syndrome, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, diabetes mellitus (type I); inflammatory conditions of, for example, the skin (e.g., psoriasis); Acute inflammatory conditions (eg endotoxemia, sepsis and septicemia, toxic shock syndrome and infectious diseases); refusal to transplant and allergy. In one embodiment, the disorder associated with TNFa is an arthritic disorder, for example, a disorder selected from one or more of rheumatoid arthritis, juvenile rheumatoid arthritis (RA) (e.g., moderate to severe rheumatoid arthritis), osteoarthritis, psoriatic arthritis or ankylosing spondylitis, juvenile idiopathic arthritis (JIA) polyarticular; or psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease and / or multiple sclerosis.

In some embodiments, the formulations include a second therapeutic agent. For example, for nanobodies of TNF, the second agent can be an anti-TNF antibody or a TNF-binding fragment thereof, wherein the second TNF antibody has an epitope specificity different from that of the binding UADS molecule to TNF of the formulation. Other non-limiting examples of agents that can be co-formulated with TNF binding UADS include, for example, a cytokine inhibitor, a growth factor inhibitor, an immunosuppressant, an anti-inflammatory agent, a metabolic inhibitor, an enzyme inhibitor, a cytotoxic and a cytostatic agent. In one embodiment, the additional agent is a standard treatment for arthritis, including, but not limited to, non-steroidal anti-inflammatory agents (NSAIDs); corticosteroids, which include prednisolone, prednisone, cortisone, and triamcinolone; and disease modifying antirheumatic drugs (FARMD), such as methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine, leflunomide (Arava®), inhibitors of tumor necrosis factor, including etanercept (Enbrel®), infliximab (Remicade®) (with or without methotrexate) and adalimumab (Humira®), anti-CD20 antibody (for example Rituxan®), soluble receptor of interleukin-1, such as anakinra (Kineret), gold, minocycline (Minocin®), penicillamine and cytotoxic agents including azathioprine, cyclophosphamide and cyclosporin. Said combination therapies can advantageously use lower dosages of the therapeutic agents administered, thus preventing possible toxicities or complications associated with the various monotherapies.

The formulations of the present invention may be in the form of a liquid solution (e.g., injectable and infusible solutions). Said compositions may be administered parenterally (eg, subcutaneous, intraperitoneal or intramuscular injection) or by inhalation. The phrases "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteric and topical administration, typically by injection and include subcutaneous or intramuscular administration, as well as intravenous, intracapsular, intraorbital administration. , intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal and infusion. In one embodiment, the formulations described herein are administered subcutaneously.

The pharmaceutical formulations are sterile and stable under the conditions of manufacture and storage. A pharmaceutical composition can also be tested to ensure that it complies with regulatory and industry standards for administration.

A pharmaceutical formulation can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high concentration of protein. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients indicated above, if necessary, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients from those enumerated above. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be carried out by including in the composition an agent that retards absorption, for example, salts of monostearate and gelatin.

In some embodiments, the characteristics of the parameters describing the formulations are described, for example, parameters that may appear on the product label. These parameters include, for example, color (typically colorless or slightly yellow or colorless to yellow), transparency (typically clear to slightly opalescent or transparent to opalescent) and viscosity (typically between about 1 and 5 cP when measured at room temperature, such as 20 ° C to 30 ° C). These parameters can be measured by methods known in the art. For example, transparency can be measured using commercially available opalescence patterns (available from, for example, Hach Company, Loveland, CO 80539).

EXAMPLES The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. These should not be construed as limiting the scope or content of the present invention in any way.

EXAMPLE 1 Stability of Freeze-dried Formulation at High Concentration of ATN-103 (duration of 6 months) A method for storing an antibody for use for, for example, therapeutic applications, is a dry powder prepared by lyophilization. Accordingly, the long-term stability of a lyophilized TNF binding formulation was studied.

Briefly, a formulation containing a humanized TNF binding nanobody (50 mg / ml), 10 mM histidine, 5% sucrose (w / v), 0.01% polysorbate-80, pH 6.0, was prepared by sterile filtration and was supplied in a 5 ml pyrogen-free glass tube vial and then lyophilized. The formulation was stored at 4 ° C, 25 ° C or 40 ° C for one month, three months and six months, then reconstituted with sterile water (USP) producing the reconstituted formulation in such a way that the formulation was a binding nanobody. TNF 100 mg / ml, histidine 20 mM, sucrose 10%, Polysorbate-80 0.02%, pH 6.0.

The stability of the liquid of high concentration was tested by biological activity, binding to Human Serum Albumin (ASH), percentage of APM and percentage of BPM by HPLC-SE, percentage of nanobody of binding to TNF and percentage of impurity without product by SDS -CE and evaluation by HPLC-CEX of relative retention time and compatibility of the elution profile with respect to the reference standard of the TNF-binding nanobody.

The lyophilized TNF-binding nanobody formulations were tested for biological activity using a test disclosed in WO 2006/122786. Figure 1 illustrates the data of said series of tests. The data were expressed with units per mg. The samples were approximately 5 - 5.5 x 106 U / mg before storage and were approximately 4.5 - 5.5 x 106 U / mg after incubation. In general, there was no substantial change in the amount of bioactivity after six months of storage in any of the samples. Therefore, the formulation is, as determined by biological activity, suitable for storage of the lyophilized formulation for at least six months.

The lyophilized TNF-binding nanobody formulations were also tested for binding activity to Human Serum Albumin (ASH). Figure 2 illustrates the data of said series of binding assays. The initial binding activity of the formulation was approximately 100% of the reference sample and did not change substantially for any of the samples during a six month trial period. Therefore, the formulation is, as determined by an ASH binding assay, suitable for storage of the lyophilized formulation for at least six months.

The percentage of APM species was tested using HPLC-SE. The percentage of APM species in the formulation before lyophilization and reconstitution was approximately 0.1% of the total protein in the formulation and also between approximately 0.1% and 0.2% in all samples stored at 4 ° C and 25 ° C (Fig. . 3). After six months of storage at 40 ° C, the formulations had approximately 0.35% APM species (Fig. 3). Therefore, there was no substantial increase in the level of APM species in samples stored at 4 ° C and 25 ° C for six months.

The percentage of BPM species was tested using HPLC-SE. The percentage of BPM species in the formulation was below the detection limit (ie 0.0%) at temperatures of 4 ° C, 25 ° C and 40 ° C for up to six months.

The percentage of nanobody binding to TNF was tested using SDS-CE. The initial percentage of the TNF-binding nanobody in the formulation was approximately 100% and did not change substantially in any of the samples during the six months of the trial period (Fig. 4).

The percentage of impurities without products was tested using SDS-CE. Insignificant impurities without product were observed by SDS-CE for the formulation at temperatures of 4 ° C, 25 ° C and 40 ° C for up to six months.

The lyophilized TNF-binding nanobody formulations were also tested for identity using HPLC-CEX. The elution profile for the formulation was comparable to reference standards at temperatures of 4 ° C, 25 ° C and 40 ° C for up to six months. The relative retention time of the designated peak remained unchanged at a standard of .00 at temperatures of 4 ° C, 25 ° C and 40 ° C for up to six months.

The effect of the addition of Polysorbate-80 on the reconstitution properties for the formulation of lyophilized TNF binding nanobody It was also tested. The addition of polysorbate 80 to the lyophilized product improves the quality of the product by improving the appearance and dissolution of the lyophilized powder as can be seen in the following table.

TABLE 1 The data described in this document show limited changes in the degradation of products as a function of storage time at various temperatures.

EXAMPLE 2 Strength of the formulation of the TNF binding nanobody for lyophilization In addition to the lyophilized formulation by applying the target lyophilization cycle (Example 1), two additional batches of pharmacological products were prepared by applying two additional cycles of "solid" lyophilization to the same formulation. The two "solid" lyophilization cycles mimic Deviations from significant procedures that could occur in a manufacturing environment. The same pharmacological product formulation was used in the solidity study as in the study of the target lyophilization cycle (control). 10 mM histidine, 5% sucrose, 0.01% Polysorbate 80, 50 mg / ml TNF binding nanobody at pH 6.0. After reconstitution, (using a volume of reconstitution diluent of approximately half of the loaded product before lyophilization) the ATN-103 formulation is as follows: 20 mM Histidine, 10% Sucrose, 0.02% Polysorbate 80 , TNF binding nanobody 100 mg / ml at pH 6.0.

The two solid lyophilization cycles that mimic significant procedural deviations are termed "high humidity" and "aggression". Figure 5 demonstrates that the formulation subjected to the solid lyophilization cycles show stability comparable to that of the target cycle (control). The solid formulation lyophilization vials were subjected to accelerated stability together with the control lyophilization cycle and analyzed by HPLC-SE.

These data demonstrate that the formulation of lyophilized ATN-103 is solid with respect to deviations from significant procedures without impact on the product.

The percentage of BP species for formulation subjected to control lyophilization and solidity cycles was tested using HPLC-SE. The percentage of BPM species by HPLC-SE for the nanobody binding to Freeze-dried TNF was below the detection limit (ie 0.0%) at t0 and 50 ° C for up to one month for all three cycles.

Lyophilization Practices In all processes, a protective layer of aluminum foil was used in front of the door and a storage height of 63 mm to minimize the radiation inside the lyophilizer. In all processes, a tray was fully loaded to conserve a homogenous charge in the lyophilizer. The stoppers were sterilized in an autoclave and dried in all vials with proteins. All vials for protein samples were rinsed with deionized water and without pyrogen. The vials and plugs that were used to load the rest of the tray were not treated.

Seed vials were prepared with the TNF-binding nanobody formulation aseptically in a bioprotective cabinet with a target of 160 mg / vial. For stability studies, the vials were loaded with 3.2 ml of freshly prepared formulation before each process (material that had not been previously lyophilized). During lyophilization, other vials were loaded with suitable buffers that were compatible with the target lyophilization cycle to maintain a homogenous charge in the lyophilizer. The lyophilization was controlled by the use of thermocouples within the protein matrix.

Differential Modulated Scanning Calorimetry (mDSC) For the mDSC, all samples were processed modulated with an amplitude of 0.5 ° C and a period of 100 seconds. For the powders after lyophilization, the samples were heated at 2 ° C / min up to 150 ° C. All powder samples were prepared using a protective glove box purged with nitrogen. For liquid samples, all temperature increases were made at 0.5 ° C / min and the temperatures were adjusted to those used in the lyophilization cycles. The final heating increase was made at 2 ° C / min to extend the glass transition. The liquid samples were prepared in the laboratory.

Moisture Analysis To assay the lyophilized samples, the Karl Fischer titration was used. The lyophilized samples were reconstituted with 3 ml of methanol.

Duplicate or triplicate injections of 500 μ ?.

As an adequate control after use, conventional water was injected 1 %.

Infrared Fourier Transform Spectroscopy (FTIR) The FTIR measured the secondary structure of the antibody in the solid powder state. A pellet containing approximately 1 mg of dry, formulated protein dispersed within 300 mg of KBr was pressed and scanned 200 times. After the data collection, the analysis involved the subtraction spectral placebo of sucrose, correction of the baseline, smoothing, secondary derivative and normalization of the area.

Stability The stability of the lyophilized antibody in the formulations was tested as a function of storage time and temperature. Samples of the lyophilized TNF-binding nanobody were tested after lyophilization, after four weeks of storage at 2 ° C-8 ° C and after two weeks and four weeks of storage at 50 ° C. The refrigerated samples were stored in a refrigerated cold room. The samples at high temperature were stored in an Imperial Lab Line Incubator set at 50 ° C. At the appropriate time points the samples were taken out of storage and allowed to warm or cool to room temperature before performing the test.

Reconstitution and Visual Appearance The vials of the lyophilized formulations of the analysis after lyophilization and storage stability analysis were visually inspected before, during and after reconstitution with 1.3 ml of sterile water for injection. The vials were inspected in a light box against a black and white background to determine the color, wet integrity particles and defects of the cake before reconstituting. After visual inspection, the lyophilized cake, the stopper and the seal are removed from the vial using a detacher. The stopper was removed and the sterile water for injection was slowly introduced into the vial using an appropriate pipette. The diluent was supplied using a swirling motion to ensure complete wetting of the cake. Once the diluent was completely distributed, the reconstitution timing was started with a conventional laboratory timer and the vial was closed again. Reconstitution was considered complete when the last part of the solid dissolved. By rolling the vial between the hands, reconstitution is facilitated. As the lyophilized cake was in the reconstruction process, observations were recorded on the state of solution dissolution such as transparency, bubble formation and foam. Once reconstitution was completed, the reconstitution time was recorded and the vials were left on the workbench for a few minutes so that the resulting solution could settle and most of the bubbles formed during reconstitution could dissipate. The reconstituted solution was then inspected in a light box against black and white background to determine the color, transparency and particles.

High Performance Chromatography by Size Exclusion (HPLC-SEC) Two microliters of net samples of TNF-binding nanobody formulation were injected into a G3000swxl column with a protective column (TosoHaas Parts No. 08541 and 08543). The mobile phase was a solution phosphate buffered saline (PBS) with 250 mM sodium chloride added. The flow rate was 0.75 ml / min and the processing time was 30 minutes. The ultraviolet absorbance was controlled at a wavelength of 280 nm. The chromatogram was integrated to separate the main peak of the TNF binding nanobody from high and low molecular weight species using the Waters Empower ™ computer program.

Ultraviolet Visible Absorbance Spectroscopy to Determine Concentration (A? BQ) Samples of the formulation had antibodies at a concentration of 100 mg / ml that were diluted to approximately 0.5 mg / ml and 0.25 mg / ml by adding 10 μ? from sample to 1990 μ? and 3990 μ? of 10 mM histidine, 5% sucrose, pH, 6.0, respectively. Two hundred microliters of the resulting solutions were placed in individual wells in a 96-well microplate along with a blank of buffer. The plate was read on a Spectramax® Plus plate reader for ultraviolet absorbance at wavelengths of 280 nm and 320 nm. By subtracting the absorbance at 320 nm from the absorbance at 280 nm and dividing by the extinction coefficient (1,405 ml / mg-cm) multiplied by the path length (1 cm), the protein concentrations of the solution in each well were determined. The appropriate dilution factor was applied and an average protein concentration was determined.

Spectroscopy of Visible Ultraviolet Absorbance for Light Dispersion (A4? O) Two hundred microliters of each sample of TNF binding nanobody to be analyzed was aliquoted into individual wells in a 96-well microplate. A blank of buffer served as control. The plate was read on a Spectramax Plus plate reader for visible absorbance at a wavelength of 420 nm.

Cycle Development Strategy A series of sequential steps (described below) was used to develop a lyophilization cycle.

Identification of the Critical Product Temperature The critical temperature of the product for a TNF binding nanobody was identified by differential modulated scanning calorimetry (mDSC). This procedure was used to identify the vitreous transition temperature of the frozen product (mDSC). A lyophilization cycle that keeps the product below this temperature during the first drying should produce an intact cake structure. It was assumed that the lowest suitable temperature was 25 ° C and therefore this temperature was generally included in the procedures designed for the conditions and formulations of the test when a formulation is developed and Methods for lyophilizing an antibody as described herein.

Execution of the Lyophilization Cycle Based on the results from the studies described above, three different lyophilization cycles were performed to examine three parameters of interest in the development of a suitable lyophilization process to prepare a lyophilized formulation suitable for storing other procedures. The first parameter examined was the control cycle, with repeated cycles from previous stability studies. All the cycles for the stability of the previous development use this cycle, since it serves as a starting point for this analysis.

The second parameter tested was the impact of not performing the second drying step, to generate lyophilized cakes with high residual moisture content. This lyophilization cycle serves as an evaluation of the sensitivity of a TNF-binding nanobody formulation with respect to high residual moisture content and can be used in the evaluation of manufacturing deviations during early clinical batches prior to lyophilization solidity studies. formal The third parameter tested was a cycle of aggressiveness.

Increasing the primary drying temperature significantly above the set point of the control cycle can significantly increase the temperature of the TNF-binding nanobody formulation product. during the first drying. This lyophilization cycle serves as an evaluation of the sensitivity of a TNF-binding nanobody formulation for the temperature of the product during lyophilization and can be used in the evaluation of manufacturing deviations during early clinical batches prior to the execution of studies of solidity of formal lyophilization.

Evaluation of Lyophilization Cycles The evaluation of the lyophilization cycles selected on TNF-binding nanobody formulations was divided into two aspects: immediate comparison based on the tests performed after lyophilization and possible long-term impact caused after incubation under accelerated conditions.

Identification of the Critical Product Temperature In the formulation product of the TNF binding nanobody it contained almost 50% protein. As such, it was predicted that the protein would dominate the physical properties of frozen and lyophilized states. Before lyophilization, the vitreous transition temperature of the frozen concentrated amorphous phase of the formulation was investigated with sub-environmental modulated Differential Scanning Calorimetry (mDSC). Based on data from the aggressive lyophilization development cycle, a temperature of the product of -12 ° C as the critical temperature below that which should be conserved during lyophilization.

EXAMPLE 3 Stability of the Liquid Formulation at High Concentration Nanobody of Union to TNF (6 months) In some cases, it is desirable to store a TNF-binding nanobody formulation in a liquid format. Accordingly, the prolonged stability of a liquid TNF-binding formulation containing a relatively high concentration of the TNF binding nanobody was studied. Briefly, a formulation containing a humanized TNF-binding nanobody (approximately 80 mg / ml), 10 mM histidine, 5% sucrose, 0.01% polysorbate 80, pH 6.0 was prepared to store the formulation by sterile filtration in stainless steel containers without pyrogen. The formulation was stored at -20 ° C or 4 ° C, for approximately three months and six months. The stability of the high concentration liquid was evaluated by biological activity, binding to Human Serum Albumin (ASH), percentage of APM and percentage of BPM by HPLC-SE, percentage of ATN-03 and percentage of impurity without product by SDS-CE and HPLC-CEX evaluation of the relative retention time and comparability of the elution profile with respect to the reference standard of the TNF-binding nanobody.

A biological activity assay was used as a stability parameter for the formulation of the high concentration liquid TNF binding nanobody. The assay was performed as described above in Example 1. The samples were stored at -20 ° C and 4 ° C for approximately three months and six months. The data were expressed as units per mg (Figure 6). The samples were approximately 6 x 106 U / mg before storage and approximately 4.5 - 5 x 106 U / mg after incubation. This essentially does not reflect changes in the bioactivity of the samples during storage. The variability of the values reflects the intrinsic variability in the assay. Because the amount of biological activity in the samples does not decrease, these data provide additional support for the suitability of the formulation for the storage of TNF binding.

Another parameter of additional stability was examined using the high-concentration liquid TNF-binding nanobody formulation: that of binding activity. In these experiments, the percentage binding activity of the formulation was determined in comparison to a control after storage at -20 ° C and 4 ° C for 6 months. The assay specifically controls the binding affinity of TNF binding to Human Serum Albumin (HSA). The initial binding activity of the formulation was approximately 100% of the reference sample and did not change substantially in any of the samples during the six-month period of the trial (Fig. 7). The measured binding activity was up to approximately 110% of the reference, which, given the error generally observed in this test, essentially reflects that there are no changes in binding activity of the samples over time and that there are no trends related to temperature in the results of test.

The percentage of APM species was tested using HPLC-SEC.

The percentage of high molecular weight species in the high concentration liquid formulation before storage was between 0.1% and 0. 5% of the total protein in the formulation and was approximately 0.1% in the samples stored at -20 ° C and approximately 0.2% of the samples stored at 4 ° C for up to six months of storage (Fig. 8). Therefore, there was no substantial increase in the level of APM species in the samples stored at -20 ° C and 4 ° C for at least six months.

The percentage of BPM species in the formulation of liquid TNF binding nanobody at high concentration was also tested in the liquid formulation of TNF-binding nanobody. The percentage of BPM species in the formulation was below the detection limit (ie 0.0%) at the temperature of -20 ° C and was approximately 0.1% in samples stored at 4 ° C for up to six months (FIG. ). Therefore, there was no substantial increase in the level of BPM species in the samples stored at -20 ° C and 4 ° C for at least six months.

The percentage of BPM species was tested using HPLC-SE. The percentage of BPM species in the liquid formulation at high concentration it was below the detection limit (ie 0.0%) at temperatures of 4 ° C, 25 ° C and 40 ° C for up to six months.

The percentage of nanobody binding to TNF was tested using SDS-CE. The initial percentage of the TNF-binding nanobody in the high-concentration liquid formulation was approximately 100% and there was no substantial change in any of the samples during the six-month test period (Fig. 10).

The percentage of impurities without product was tested using SDS-CE. Impurity without negligible product was observed by SDS-CE for the formulation of liquid TNF binding nanobody at high concentration at temperatures of -20 ° C and 4 ° C for six months.

High concentration liquid formulations were also tested for identification using HPLC-CEX. HPLC-CEX was used as an identity assay. The elution profile for TNF binding of the high concentration liquid formulation was comparable to the reference standard at temperatures of -20 ° C and 4 ° C for up to six months. The relative retention time of designed peaks did not change to a pattern of 1.00 at temperatures of -20 ° C and 4 ° C for up to six months.

The data described in this document show limited changes in degradation products as a function of shelf life at various temperatures.

EXAMPLE 4 Stability of the High Concentration Liquid Formulation of the TNF-binding nanobody in a liquid syringe previously charged (12 months) The stability of a high concentration liquid of TNF binding nanobody in a syringe loaded in the following formulation: 10 mM histidine, 5% sucrose, 0.01% polysorbate 80, approximately 80 mg / ml nanobody of TNF binding, at pH 6.0, was assayed by percentage of APM and percentage of BPM by HPLC-SE and percentage of acidic and basic species by HPLC-CEX and evaluation of the relative retention time and comparability of the elution profile with respect to the reference pattern of TNF binding nanobody. The formulation was stored at 4 ° C for twelve months, at 25 ° C for three months and at 40 ° C for two months.

At the initial time point, there was approximately 0.7% of APM species. After twelve months at 4 ° C there was a minimum increase of about 0.8% of APM species. After three months at 25 ° C, the APM species increased approximately 1.8%. After two months at 40 ° C, the APM species increased over time to approximately 27% (Fig. 11).

At the initial time point, there was 0.1% of BPM species. After twelve months at 4 ° C there was a minimum increase to 0.25% of BPM species. After three months at 25 ° C, there was a small increase to approximately 0.5% BPM. After two months at 40 ° C, the degradation increased over time to approximately 1.4% of BPM species (Fig. 12).

At the initial time point, there were approximately 6% acid species. After twelve months at 4 ° C there were approximately 7.5% acid species. After three months at 25 ° C, there was approximately 7.3% acidic species, increasing the acid species over time. After two months at 40 ° C, the acid species increased over time to approximately 8.3% (Fig. 13).

At the initial time point, there was approximately 1.7% of basic species. After twelve months at 4 ° C there were approximately 2.9% of basic species. After three months at 25 ° C, there were approximately 2.9% of basic species, increasing the basic species over time. After two months at 40 ° C, the basic species increased over time to approximately 27% (Fig. 14).

The relative retention times and elution profiles of all samples were comparable to the reference standard of the TNF binding nanobody.

The data show limited changes in degradation products as a function of storage time at 4 ° C and 25 ° C, indicating that the formulation is suitable as a liquid in a syringe previously loaded. Some perceptible changes were seen in the degradation products at 40 ° C, which is a state of stress for a liquid.

EXAMPLE 5 Stability of ATN-103 High Concentration Liquid - Other Formulations (identification of other stabilizing and destabilizing excipients) In order to explore possible excipients for a liquid TNF-binding nanobody formulation, the stability of other liquid formulations of high concentration TNF-binding nanobody was examined. A complementary work was performed using various excipients to provide additional stability and prepare the isotonic formulation (suitable for injection in human subjects). The concentration of TNF-binding nanobody varies from 88 mg / ml to 100 mg / ml.

The formulations examined were: 1. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 100 mM Arginine (base), pH 5.8. 2. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 55 mM NaCl, pH 6.1. 3. 10 mM histidine, 5% sucrose, 0.01% polyisobate-80, 55 mM Arginine (base), pH 6.1. 4. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 100 mM glycine, pH 6.0. 5. 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, 100 mM methionine, pH 6.0. 6. 10 mM histidine, 8% sucrose, 0.01% polysorbate-80, pH 6.0 CTL: 10 mM histidine, 5% sucrose, 0.01% polysorbate-80, pH 6.0.

The properties of the initial solution were analyzed for pH, osmolarity, concentration, turbidity and viscosity. All formulations resulted in isotonic solutions and showed acceptable transparency by measurement by A455 and low viscosity (from 2.4 cP to 3.1 cP), which shows viability of previously loaded syringe and autoinjector.

TABLE 2 The stability of the high concentration liquid was evaluated by percentage of APM and percentage of BPM by HPLC-SE. These materials were subjected to stability at 5 ° C, 25 ° C and 40 ° C for 3 months. Data from 2 weeks at 40 ° C are shown in Figure 15.

Some perceptible changes were seen in the degradation products at 40 ° C, which is a state of stress for a liquid. Accelerated brief stability (2 weeks at 40 ° C) shows that formulations 4, 5 and 6 offer comparable or improved stability with respect to control (10 mM histidine, 5% sucrose, 0.01% polysorbate-80, pH 6 , 0). Formulations 1, 2 and 3 appear to have a negative impact on stability.

The data shows that the increased glycine, methionine and sucrose are stabilizing with respect to liquid formulations of high concentration TNF-binding nanobody. The data show that the arginine base, arginine hydrochloride and sodium chloride can be destabilizing to liquid formulations of TNF-binding nanobody at high concentration in some conditions.

EXAMPLE 6 Stability of the TNF-binding nanobody of the Liquid Formulation at High Concentration, of short duration (2 weeks duration), Histidine and Tris buffers The stability of the TNF-binding nanobody as a liquid is illustrated in the following Figures 16-19. Two formulations were examined: ATN-103 at 118 mg / ml in 20 mM Histidine, 5% Sucrose, pH 6 and ATN-103 at 117 mg / ml in 20 mM Tris, 5% sucrose, pH 7.2. The stability of the formulations was tested by percentage of APM and percentage of BPM by HPLC-SE and percentage of acidic species and percentage of basic species by HPLC-CEX. The data show limited changes in degradation products, as a function of storage time at 4 ° C. Some perceptible changes were seen in the degradation products at 40 ° C, which is a state of stress for a liquid. The data shows that the stability of the TNF-binding nanobody in Histidine and Tris buffers is essentially similar under these formulation conditions, yielding slightly more favorably with histidine (slightly less BPM). The pre-formulation activities would finally determine that the high pH (7 or higher) results in a higher degree of BPM formation, explaining the advantage observed below.

EXAMPLE 7 Stability of High Concentration Liquid Formulation of TNF-binding nanobody: Evaluation of interfacial stress (freezing / thawing) Figures 20-23 demonstrate the formulation stability of nanobody binding to liquid TNF at about 80 mg / ml in 10 mM Histidine, 5% Sucrose, 0.01% Polysorbate 80, pH 6.0. The evaluation was based on HPLC by size exclusion, turbidity and concentration titration followed by multiple freeze / thaw cycles of -80 ° C and 37 ° C.

The data show limited changes in stability as a function of multiple cycling of freeze / thaw at -80 ° C and 37 ° C.

EXAMPLE 8 Stability of High Concentration Liguida Formulation of TNF-binding nanobody: Assessment of short term thermal stress possibly found in manufacturing procedures Figure 24 demonstrates that the liquid TNF binding nanobody is solid with respect to short duration thermal stress that could possibly be encountered during the preparation procedures of the pharmacological substances and pharmacological products. The high concentration liquid was studied in 10 mM Histidine, 5% Sucrose, 0.01% Polysorbate 80, pH 6.0 at approximately 80 mg / ml and 50 mg / ml. The evaluation was based on the percentage of APM and BP percentage by size exclusion HPLC, after exposure for 8 hours at 40 ° C, 7 days at 25 ° C and 29 days at 5 ° C. The data show limited changes in aggregates as a function of storage time at 5 ° C and 25 ° C. Some changes were observed in the aggregates at 40 ° C, which is a state of stress for a liquid.

The percentage of APM species by HPLC-SE for the high concentration liquid of the TNF binding nanobody was below the detection limit (ie 0.0%) at the indicated temperatures and durations.

EXAMPLE 9 Stability of the Low Concentration Liquid Formulation of ATN-103: Optimal pH Rating and Formulation Figures 25-28 demonstrate the stability of a liquid TNF-binding nanobody formulation at low concentration (approximately 1 mg / ml) buffered at pH 5.5, 6.0, 6.5 and 7.0. The stability of the low concentration liquid TNF-binding nanobody was examined as a function of formulation and pH in response to stress such as exposure to 40 ° C temperature (Figures 25 and 26), agitation (Fig. 28) and freezing / thawing. Four pH were evaluated for each of the three following formulations: 10 mM histidine, 5% sucrose, 0.01% Tween-80; 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginine HCI; and 10 mM histidine, 5% sucrose, 0.01% Tween-80; 75 mM sodium chloride. In this data set, Tween-80 was used as a synonym for Polysorbate 80. The study samples were evaluated using HPLC-SE and UV (both for the concentration and for the turbidity - measured by A455).

Codes of the Figures: HST: 10 mM histidine, 5% sucrose, 0.01% Tween-80 HSTA: 10 mM histidine, 5% sucrose, 0.01% Tween-80, 150 mM arginine HCI HSTS: 10 mM histidine, 5% sucrose, 0.01% Tween-80, 75 mM sodium chloride.

The results show that the pH range of 5.5 - 7.0 is adequate for the formulation. The data show that under these same conditions, pH 7.0 may show some detrimental effects (increase of low molecular weight species). The data show that there is no significant benefit in terms of the addition of HCI arginine or sodium chloride to the drug substance formulation and in some cases it can be destabilizing.

Figure 27 shows the percentage of APM species by HPLC-SE for the TNF-binding nanobody after storage at 4 ° C, in which no essential change was observed after 4 weeks. The percentage of BPM species by HPLC-SE for the TNF binding nanobody at low concentration was lower than the detection limit (ie 0.0%) at 4 ° C for all conditions of the tested solution. No significant changes were observed in AMP or BPM species by HPLC-SE or UV A280 or A455 as a result of multiple freeze / thaw cycles.

EXAMPLE 10 TNF binding nanobody Liquid at Low Concentration: Evaluation of the Effect of Agitation as a Function of pH and Formulation Data are also presented demonstrating that the TNF-binding nanobody is sensitive to agitation at 300 rpm for 4 hours (at 15 ° C) during this pH range (Fig. 28). Formulations containing sodium chloride and arginine are especially sensitive to agitation. The formulation of histidine, sucrose, tween-80 showed the lowest degradation of low molecular weight in each pH group. The formulation of histidine, sucrose and tween-80 at pH 6.0 and 7.0 showed the lowest APM degradation.

The UV absorbance of the low concentration TNF-binding nanobody after agitation was monitored at 280 nm (to control the concentration) and 455 nm (to control turbidity). No significant changes were observed as a result of the agitation.

The low concentration TNF binding nanobody solutions were examined after multiple freeze / thaw cycles by HPLC-SE and UV analysis at 280 nm (to control the concentration) and 455 nm (to control turbidity). No significant changes were observed in HPLC-SE or UV A280 or A455 as a result of multiple freeze / thaw cycles.

EXAMPLE 11 Stability of nanobody of union to TNF of Liquid Formulation at High Concentration, of short duration (2 weeks of duration), examining tonicity adjusting agents The stability of the TNF binding nanobody as a liquid is illustrated in the following: Five formulations were examined as shown in Figures 31 and 32 referred to herein as HST, HSGT, HSG T, HSorb and Control. Each of the formulations examined is described below.

Fig. 31 and 32 Formulations HST 100 mg / ml of TNF binding nanobody, 20 mM histidine, 8% sucrose, 0.01% polysorbate 80 HSGT 100 mg / ml of TNF binding nanobody, 20 mM histidine, 5% sucrose, 80 mM glycine, 0.01% polysorbate 80 HSGMT 100 mg / ml of TNF-binding nanobody, 20 mM histidine, 5% sucrose, 80 mM glycine, 10 mM methionine, 0.01% polysorbate 80 HSorb 100 mg / ml of TNF binding nanobody, 20 mM histidine, 5% sorbitol 100 mg / ml control of TNF-binding nanobody, 20 mM histidine, 5% sucrose The formulations were stored as a liquid for two weeks at 4 ° C and 40 ° C (stress state) in polypropylene tubes and in syringes previously loaded with cyclic olefin copolymer with a plunger rubber.

The stability of the formulations was evaluated by percentage of APM and percentage of BPM by HPLC-SE as represented in Figures 31 and 32. The data show limited changes in the products of degradation as a function of storage time at 4 ° C. For the samples shown in Figure 32, no BPM was detected at the time point initial or after two weeks at 4 ° C. Only BPM was detected in the samples at 40 ° C (with stress). The data show that the five formulations show comparable changes in the products of degradation as a function of storage time in the state of Stress at 40 ° C. Therefore, the data shows that all formulations are suitable for liquid dosage forms.

EXAMPLE 12 Stability of TNF binding nanobody in Liquid Formulation at Low Concentration and High Concentration, confirming the target formulation and examining primary packaging containers The stability of the TNF binding nanobody as a liquid is illustrated below: Three formulations were examined: (a) 10 mg / ml of TNF binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01% polysorbate 80; (b) 50 mg / ml of TNF binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01% polysorbate 80; (c) 100 mg / ml of TNF binding nanobody, 20 mM histidine, 7.5% sucrose, 0.01% polysorbate 80; The formulation was prepared in the following primary packaging containers: (a) syringe of pharmaceutical quality of pre-loadable glass of Type I of a supplier and a plunger of gray silicone rubber West 4432 (b) syringe of pharmaceutical quality of pre-loadable glass of Type I of a second supplier and a plunger of gray rubber of silicone West 4432 (c) pre-charged cyclic olefin copolymer and a West 4432 gray silicone rubber plunger.

The formulations were analyzed at t = 0 and found to be satisfactory. The formulation had been stored at 4 ° C, 25 ° C and 40 ° C for three months.

Equivalents All references cited herein are incorporated herein by reference in their entirety and for all purposes as indicated individually and specifically for each publication, patent or individual patent application incorporated by reference in its entirety for all purposes. .

The scope of the present invention is not limited to the specific embodiments described herein. In fact, various modifications of the invention in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. It is intended that said modifications be included within the scope of the appended claims.

Claims (1)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A formulation comprising: (a) a nanobody molecule binding to TNF at a concentration of about 10 mg / ml to about 250 mg / ml; (b) a lyoprotectant selected from sucrose, sorbitol or trehalose at a concentration of about 5% to about 10%; (c) a surfactant selected from polysorbate-80 or poloxamer-188 at a concentration of about 0.01% to 0.6%; and (d) a buffer chosen between histidine cap at a concentration of about 10 to about 20 mM or a Tris buffer at a concentration of about 20 mM such that the pH of the formulation is from about 5.0 to 7.5, wherein the TNF-binding nanobody molecule in the formulation retains at least about 70% of its binding activity after storage for at least three months at 4 ° C. 2. - The formulation according to claim 1, further characterized in that it has: (i) less than 5% of high molecular weight species (MPA) after storage for at least 12 months at 4 ° C; (i) less than 5% of low molecular weight species (BPM) after storage for at least 12 months at 4 ° C; (iii) less than 10% acidic species after storage for at least 12 months at 4 ° C; and / or (iv) less than 5% of basic species after storage for at least 12 months at 4 ° C. 3. - The formulation according to claim 1 or 2, further characterized in that it is in the form of a liquid bulk storage, freeze-dried, reconstituted or frozen lyophilized. 4. - The formulation according to any of claims 1-3, further characterized in that it is a liquid or lyophilized formulation comprising: (a) a nanobody molecule binding to TNF at a concentration of about 10 mg / ml to about 130 mg / ml; (b) sucrose at a concentration of about 5% to about 10%; (c) polysorbate-80 at a concentration of about 0.01% -0.02%; and (d) a buffer selected from the group consisting of histidine buffer at a concentration of about 10 to about 20 mM, such that the pH of the formulation is from about 5.0 to 7.5. 5. - The formulation according to any of claims 1-3, further characterized in that it is a bulk storage formulation comprising: (a) a nanobody molecule binding to TNF at a concentration of about 80 mg / ml to about 280 mg / ml; (b) sucrose at a concentration of about 5% to about 10%; (c) polysorbate-80 at a concentration of about 0.01% to 0.02%; and (d) a buffer selected from the group consisting of histidine buffer at a concentration of about 10 to about 20 mM, so that the pH of the formulation is from about 5.0 to 7.5; wherein at least 100 liters of the formulation are stored under conditions below freezing. 6. - The formulation according to any of claims 1-5, further characterized in that the pH of the formulation is selected from the group consisting of 5, 5.5, 5.8-6.1, 6.0, 6.1, 6.5 and 7. 7. - The formulation according to any of claims 1-6, further characterized in that the sucrose, sorbitol or trehalose is at a concentration of about 5%, about 7.5% or about 10%. 8. - The formulation according to any of claims 1-7, further characterized in that the TNF-binding nanobody molecule is a single-chain polypeptide comprising one or more single domain molecules. 9. - The formulation according to claim 8, further characterized in that the TNF-binding nanobody molecule is monovalent or multivalent. 10. - The formulation according to claim 8, further characterized in that the TNF-binding nanobody molecule is monospecific or multispecific. 1 - The formulation according to claim 8, further characterized in that one or more single domain molecules are grafted to CDR, humanized, camelized, deimmunized or selected by phage display. 12. - The formulation according to claim 8, further characterized in that the TNF-binding nanobody molecule is a single-chain fusion polypeptide comprising one or more single-domain molecules that bind to tumor necrosis factor-a (TNFa) and a single-domain molecule that binds to human serum albumin protein (HSA). 13. - The formulation according to any of claims 1-7, further characterized in that the TNF-binding nanobody molecule comprises the amino acid sequence of SEQ ID NO: 1 or at least 90% identical amino acid sequence thereto. 14. - The formulation according to any of claims 1-7, further characterized in that at least one of the single domain molecules of the TNF-binding nanobody molecule comprises three CDRs having the amino acid sequence: DYWMY (SEQ ID NO. : 2) (CDR1), EINTNGLITKYPDSVKG (SEQ ID NO: 3) (CDR2) and SPSGFN (SEQ ID NO: 4) (CDR3), or having a CDR that differs in a conservative amino acid substitution of one of said CDRs. 15. - The formulation according to any of claims 1-7, further characterized in that at least one of the single domain molecules of the TNF-binding nanobody molecule comprises a variable region having the amino acid sequence of about 1 to 15 amino acids of SEQ ID NO: 1 or a variable region that differs by up to 10 amino acids from said variable region. 16. - The formulation according to any of claims 1-7, further characterized in that the TNF-binding nanobody molecule further comprises at least one single domain molecule that binds to ASH and comprises three CDRs having the amino acid sequence: SFGMS (SEQ ID NO: 5) (CDR1), SISGSGSDTLYADSVKG (SEQ ID NO: 6) (CDR2) and GGSLSR (SEQ ID NO: 7) (CDR3) or having a CDR that differs in a conservative amino acid substitution of a of said CDRs. 17. - The formulation according to any of claims 1-7, further characterized in that at least one of the single domain molecules of the TNF-binding nanobody molecule binds to ASH and comprises a variable region having the amino acid sequence from about 125 to 239 amino acids of SEQ ID NO: 1, or a variable region that differs by up to 10 amino acids from said variable region. 18. - A method or process for preparing a formulation of a TNF binding nanobody, comprising: expressing the TNF binding nanobody in a cell culture; purifying the TNF binding nanobody by passing the TNF binding nanobody through at least one chromatography purification step, or an ultrafiltration / diafiltration step; adjust the concentration of the TNF binding nanobody approximately 10 to 250 mg / ml in a formulation containing sucrose at a concentration of about 5% to about 10%; polysorbate-80 at a concentration of about 0.01%, 0.02% and a histidine buffer at a concentration of about 10 to about 20 mM or a Tris buffer at a concentration of about 20 mM, so that the pH of the formulation is from about 5 to 7.5. 19. - A process for preparing a reconstituted formulation containing a TNF-binding nanobody molecule, comprising: lyophilizing a mixture of a TNF-binding nanobody molecule and a lyoprotectant, a surfactant and a buffer, thereby forming a mixture lyophilized and reconstitute the lyophilized mixture in a diluent, thereby preparing the formulation, wherein the reconstituted formulation comprises (a) a nanobody molecule binding to TNF at a concentration of about 10 mg / ml to about 130 mg / ml; (b) a lyoprotectant selected from the group consisting of sucrose or trehalose at a concentration of about 5% to about 10%; (c) polysorbate-80 as the surfactant at a concentration of about 0.01% to 0.02% and (d) a histidine buffer at a concentration of about 10 to about 20 mM or a Tris buffer at a concentration of about 20 mM so that the pH of the formulation is about 5.0 to 7.5. 20. - A kit or article of manufacture comprising a package containing the formulation of any of claims 1-17 and instructions for its use. 21. The kit or article of manufacture according to claim 20, further characterized in that the formulation is presented in a vial or in an injectable syringe. 22. The kit or article of manufacture according to claim 20, further characterized in that the formulation is presented in an injectable syringe previously loaded. 23. The kit or article of manufacture according to claim 21, further characterized in that the syringe or a vial is composed of glass, plastic or a polymeric material selected from a cyclic olefin polymer or copolymer. 24. - The use of the formulation of any of claims 1-17, for preparing a medicament for treating or preventing a disorder related to TNF in a subject. 25. - The use as claimed in claim 24, wherein the disorder related to TNF is an inflammatory or autoimmune disorder. 26. - The use as claimed in claim 24, wherein the TNF-related disorder is selected from rheumatoid arthritis (RA), arthritic conditions (e.g., psoriatic arthritis, juvenile idiopathic arthritis (JIA) polyarticular, ankylosing spondylitis (EA) ), psoriasis, colitis ulcerative, Crohn's disease, inflammatory bowel disease or multiple sclerosis. 27. - A method for analyzing a manufacturing process, comprising: providing a sample of the formulation of any of claims 1-17; evaluate a parameter of the selected formulation of color, clarity, viscosity or a quantity of one or more APM, acidic or basic BPM species, determine if the parameter fulfills a preselected criterion, analyzing in this way the process. 28. - The method according to claim 27, further characterized in that it further comprises comparing two or more formulations of a sample in a control or monitoring procedure of variation from batch to batch or comparing the sample with respect to a reference standard. 29. - The method according to claim 28, further characterized in that it further comprises classifying, selecting, accepting or discarding, distributing or retaining, processing in a pharmacological product, transporting, moving to a different location, formulating, marking, packaging the formulation, base to the comparison. 30. - The method according to claim 29, further characterized in that it further comprises providing a record that includes data related to the evaluated parameter of the formulation and optionally includes an identifier for a batch of the formulation; Submit said record to the person making decisions; optionally, receive a statement from the person making decisions; optionally, decide whether the formulation lot is distributed or put on sale based on the communication of the person making decisions.